Engineered Antibody-Stress Protein Fusions

ABSTRACT

Provided are fusion polypeptides comprising an engineered antibody and a stress protein that bind to antigens with high affinity, are highly immunogenic, exhibit MHC class I priming and are able to be produced in non-mammalian cells, such as  E. coli.

RELATED APPLICATIONS

This application is a continuation-in-part application ofPCT/US07/061554, filed Feb. 2, 2007, which claims priority to U.S.application Ser. No. 60/764,620 filed Feb. 2, 2006. The contents of eachof these applications is expressly incorporated herein by reference.

GOVERNMENT SUPPORT

The subject invention was made in part with government support underDepartment of State Grant S-LMAQM-04-GR-164, awarded by the AcceleratedDrug and Vaccine Development with Former Soviet Union Institutions inSupport of the U.S. Department of State BioIndustry Initiative.Accordingly, the U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Classical monoclonal antibodies are currently produced in mammaliancells. Drawbacks of this method of production include the difficulty ofproducing and selecting appropriate clones, and the expense of culturingmammalian cells. The “next generation” of monoclonal antibodies arebeing engineered in E. coli. Recently, microbial expression of V_(H) andV_(L) domains tethered together by polypeptide linkers has created thecapability of generating engineered “mini-antibodies.” These mini-bodiescan be generated in E. coli in a virtually combinatorial fashion. Theseartificially created Fab or single chain Fv (scFv) can be linkedtogether to form multimers, e.g., diabodies, triabodies and tetrabodies.Although they are capable of binding to antigens with almostantibody-like efficiency, these engineered, Fc deficient mini-antibodieslack the ability to interact with antigen presenting cells and arepoorly immunogenic.

Existing solutions to the lack of immunogenicity of engineeredantibodies involve directing one of the antigen binding sites to binddirectly with immune cells. This brings them in apposition, but does notresult in the same MHC class I priming as would be observed for amonoclonal antibody.

SUMMARY OF THE INVENTION

In one aspect, provided are fusions of an engineered antibody, such as aFab or scFv, with a stress protein, such as HSP70. Stress proteins arevery efficient at presenting antigens to antigen presenting cells andprovoking a T cell response. They have been particularly effective ateliciting cell mediated immune and humoral immune responses by thispathway.

Thus, the fusion molecules bind to antigens with high affinity, arehighly immunogenic, exhibit MHC class I priming, provoke a T cellresponse and are able to be produced in non-mammalian systems such as E.coli. The fusion molecules are thus suitable for use as highlyimmunogenic vaccines for the prevention or treatment of infectious,inflammatory, autoimmune, or malignant disease.

Accordingly, provided are fusion polypeptides comprising at least oneengineered antibody and at least one stress protein. These engineeredantibodies comprising the fusion polypeptides may be multivalent, i.e.,they may be bivalent, trivalent, tetravalent, pentavalent, etc. Further,they may be monospecific or multispecific.

Also provided are nucleic acids and vectors encoding the engineeredantibody-stress protein fusion polypeptides, host cells comprising thenucleic acids and vectors and methods for producing the engineeredantibody-stress protein fusion polypeptides. Antigen combining sites orengineered antibody fragments can be created quickly and with highaffinity, and may be inexpensively fused to a stress protein.

Further provided are pharmaceutical and vaccine compositions comprisingthe subject engineered antibody-stress protein fusion polypeptides. Suchcompositions may further comprise an adjuvant or other agent. Alsoprovided are methods of preventing or treating infectious, inflammatory,autoimmune or malignant disease in a patient comprising administering toa patient in need thereof, an effective amount of any one of theaforementioned compositions.

Kits for the practice of the methods are also described herein.

Other features and advantages will be apparent from the followingdetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary engineered antibody-stress protein fusionpolypeptide comprising a tetravalent Tandab (engineered antibody) andHSP70 (stress protein).

FIG. 2 depicts the full-length polypeptide sequences of HSP70 fromMycobacterium tuberculosis HSP70 and Mycobacterium bovus HSP70,respectively.

FIG. 3 depicts the results of 12% PAAG as described in Example 1, allproteins loaded at 4 μg/track under denaturing (DTT) condition.

FIG. 4. OD_(450nm). The antigens for coating were taken at theconcentration 1 μg/ml in PBS. The primary antibodies were incubated inPBS with 0.2% BSA and 0.05% Tween 20 at serial dilutions. Secondaryanti-mouse antibodies conjugated with HRP were used.

FIG. 5 illustrates modification of the pET-45b(+) expression vector. A)Schematic of the insertion of the SfiI restriction site. B) Finalmultiple cloning sites of the modified pET-45b(+) vector.

FIG. 6 shows introduction of NotI and XhoI restriction sites intoMTBhsp70. Using primers overlapping the N- and C-terminals of MTBhsp70 aNotI site and an XhoI site were introduced by PCR. The resulting PCRproduct is shown in lane 1.

FIG. 7 shows digestion of the amplified MTBhsp70 with NotI and XhoIyielding 2 bands as shown on the gel picture. Sequencing analysesestablished the presence of internal NotI and SfiI sites.

FIG. 8 shows removal of NotI and SfiI sites by using PCR based sitedirected mutagenesis. Using modified primers containing the desiredmutations, two overlapping PCR products were generated. These wereextended by PCR in absence of primers followed by the addition ofspecific primers.

FIG. 9 shows introduction of the Ovalbumin peptide (residues 254-264) atthe N-terminal of MTBhsp70. A synthetic linker coding for the Ovalbuminpeptide SIINFEKL was digested with SfiI and NotI. A pET-45b(+) MTBhsp70plasmid was cut with SfiI and NotI. Both components were ligated and theligation product was used to transform competent bacteria. Ampicillinresistant colonies were picked and screened by sequencing.

FIG. 10 shows partial sequencing of the N-terminal region ofOva₂₅₇₋₂₆₄-MTBhsp70.

FIG. 11 shows an MTB HSP 70 fusion with an antibody.

DETAILED DESCRIPTION

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are defined here.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

The term “administering” includes any method of delivery of a compoundof the present invention, including but not limited to, a pharmaceuticalcomposition or therapeutic agent, into a subject's system or to aparticular region in or on a subject. The phrases “systemicadministration,” “administered systemically,” “peripheraladministration” and “administered peripherally” as used herein mean theadministration of a compound, drug or other material other than directlyinto the central nervous system, such that it enters the patient'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration. “Parenteral administration” and“administered parenterally” means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof, amino acid analogs having variant side chains; and allstereoisomers of any of the foregoing. The names of the natural aminoacids are abbreviated herein in accordance with the recommendations ofIUPAC-IUB.

The term “antibody” refers to an immunoglobulin, derivatives thereofwhich maintain specific binding ability, and proteins having a bindingdomain which is homologous or largely homologous to an immunoglobulinbinding domain. These proteins may be derived from natural sources, orpartly or wholly synthetically produced. An antibody may be monoclonalor polyclonal. The antibody may be a member of any immunoglobulin classfrom any species, including any of the human classes: IgG, IgM, IgA,IgD, and IgE. In exemplary embodiments, antibodies used with the methodsand compositions described herein are derivatives of the IgG class.

The term “antibody fragment” refers to any derivative of an antibodywhich is less than full-length. In exemplary embodiments, the antibodyfragment retains at least a significant portion of the full-lengthantibody's specific binding ability. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv, dsFvdiabody, Fc, and Fd fragments. The antibody fragment may be produced byany means. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody, it may berecombinantly produced from a gene encoding the partial antibodysequence, or it may be wholly or partially synthetically produced. Theantibody fragment may optionally be a single chain antibody fragment.Alternatively, the fragment may comprise multiple chains which arelinked together, for instance, by disulfide linkages. The fragment mayalso optionally be a multimolecular complex. A functional antibodyfragment will typically comprise at least about 50 amino acids and moretypically will comprise at least about 200 amino acids.

The term “antigen binding site” refers to a region of an antibody thatspecifically binds an epitope on an antigen.

The terms “comprise” and “comprising” is used in the inclusive, opensense, meaning that additional elements may be included.

The term “effective amount” refers to that amount of a compound,material, or composition which is sufficient to effect a desired result.An effective amount of a compound can be administered in one or moreadministrations.

The term “engineered antibody” refers to a recombinant molecule thatcomprises at least an antibody fragment comprising an antigen bindingsite derived from the variable domain of the heavy chain and/or lightchain of an antibody and may optionally comprise the entire or part ofthe variable and/or constant domains of an antibody from any of the Igclasses (for example IgA, IgD, IgE, IgG, IgM and IgY).

The term “epitope” refers to the region of an antigen to which anantibody binds preferentially and specifically. A monoclonal antibodybinds preferentially to a single specific epitope of a molecule that canbe molecularly defined. In the present invention, multiple epitopes canbe recognized by a multispecific antibody.

A “fusion protein” or “fusion polypeptide” refers to a hybridpolypeptide which comprises polypeptide portions from at least twodifferent polypeptides. The portions may be from proteins of the sameorganism, in which case the fusion protein is said to be “intraspecies”,“intragenic”, etc. In various embodiments, the fusion polypeptide maycomprise one or more amino acid sequences linked to a first polypeptide.In the case where more than one amino acid sequence is fused to a firstpolypeptide, the fusion sequences may be multiple copies of the samesequence, or alternatively, may be different amino acid sequences. Afirst polypeptide may be fused to the N-terminus, the C-terminus, or theN- and C-terminus of a second polypeptide. Furthermore, a firstpolypeptide may be inserted within the sequence of a second polypeptide.

The term “Fab fragment” refers to a fragment of an antibody comprisingan antigen-binding site generated by cleavage of the antibody with theenzyme papain, which cuts at the hinge region N-terminally to theinter-H-chain disulfide bond and generates two Fab fragments from oneantibody molecule.

The term “F(ab′)2 fragment” refers to a fragment of an antibodycontaining two antigen-binding sites, generated by cleavage of theantibody molecule with the enzyme pepsin which cuts at the hinge regionC-terminally to the inter-H-chain disulfide bond.

The term “Fc fragment” refers to the fragment of an antibody comprisingthe constant domain of its heavy chain.

The term “Fv fragment” refers to the fragment of an antibody comprisingthe variable domains of its heavy chain and light chain.

“Gene construct” refers to a nucleic acid, such as a vector, plasmid,viral genome or the like which includes a “coding sequence” for apolypeptide or which is otherwise transcribable to a biologically activeRNA (e.g., antisense, decoy, ribozyme, etc), may be transfected intocells, e.g. in certain embodiments mammalian cells, and may causeexpression of the coding sequence in cells transfected with theconstruct. The gene construct may include one or more regulatoryelements operably linked to the coding sequence, as well as intronicsequences, polyadenylation sites, origins of replication, marker genes,etc.

“Host cell” refers to a cell that may be transduced with a specifiedtransfer vector. The cell is optionally selected from in vitro cellssuch as those derived from cell culture, ex vivo cells, such as thosederived from an organism, and in vivo cells, such as those in anorganism. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The term “including” is used herein to mean “including but not limitedto”. “Including” and “including but not limited to” are usedinterchangeably.

The term “immunogenic” refers to the ability of a substance to elicit animmune response. An “immunogenic composition” or “immunogenic substance”is a composition or substance which elicits an immune response. An“immune response” refers to the reaction of a subject to the presence ofan antigen, which may include at least one of the following: makingantibodies, developing immunity, developing hypersensitivity to theantigen, and developing tolerance.

The term “isolated polypeptide” refers to a polypeptide, which may beprepared from recombinant DNA or RNA, or be of synthetic origin, somecombination thereof, or which may be a naturally-occurring polypeptide,which (1) is not associated with proteins with which it is normallyassociated in nature, (2) is isolated from the cell in which it normallyoccurs, (3) is essentially free of other proteins from the same cellularsource, (4) is expressed by a cell from a different species, or (5) doesnot occur in nature.

The term “isolated nucleic acid” refers to a polynucleotide of genomic,cDNA, synthetic, or natural origin or some combination thereof, which(1) is not associated with the cell in which the “isolated nucleic acid”is found in nature, or (2) is operably linked to a polynucleotide towhich it is not linked in nature.

The term “linker” is art-recognized and refers to a molecule or group ofmolecules connecting two compounds, such as two polypeptides. The linkermay be comprised of a single linking molecule or may comprise a linkingmolecule and a spacer molecule, intended to separate the linkingmolecule and a compound by a specific distance.

The term “multivalent antibody” refers to an antibody or engineeredantibody comprising more than one antigen recognition site. For example,a “bivalent” antibody has two antigen recognition sites, whereas a“tetravalent” antibody has four antigen recognition sites. The terms“monospecific”, “bispecific”, “trispecific”, “tetraspecific”, etc. referto the number of different antigen recognition site specificities (asopposed to the number of antigen recognition sites) present in amultivalent antibody. For example, a “monospecific” antibody's antigenrecognition sites all bind the same epitope. A “bispecific” antibody hasat least one antigen recognition site that binds a first epitope and atleast one antigen recognition site that binds a second epitope that isdifferent from the first epitope. A “multivalent monospecific” antibodyhas multiple antigen recognition sites that all bind the same epitope. A“multivalent bispecific” antibody has multiple antigen recognitionsites, some number of which bind a first epitope and some number ofwhich bind a second epitope that is different from the first epitope.

The term “nucleic acid” refers to a polymeric form of nucleotides,either ribonucleotides or deoxynucleotides or a modified form of eithertype of nucleotide. The terms should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single-stranded(such as sense or antisense) and double-stranded polynucleotides.

“Protein” (if single-chain), “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product, e.g., as may beencoded by a coding sequence. When referring to “polypeptide” herein, aperson of skill in the art will recognize that a protein can be usedinstead, unless the context clearly indicates otherwise. A “protein” mayalso refer to an association of one or more polypeptides. By “geneproduct” is meant a molecule that is produced as a result oftranscription of a gene. Gene products include RNA molecules transcribedfrom a gene, as well as proteins translated from such transcripts.

The terms “polypeptide fragment” or “fragment”, when used in referenceto a particular polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thatof the reference polypeptide. Such deletions may occur at theamino-terminus or carboxy-terminus of the reference polypeptide, oralternatively both. Fragments typically are at least about 5, 6, 8 or 10amino acids long, at least about 14 amino acids long, at least about 20,30, 40 or 50 amino acids long, at least about 75 amino acids long, or atleast about 100, 150, 200, 300, 500 or more amino acids long. A fragmentcan retain one or more of the biological activities of the referencepolypeptide. In various embodiments, a fragment may comprise anenzymatic activity and/or an interaction site of the referencepolypeptide. In another embodiment, a fragment may have immunogenicproperties.

A “patient” or “subject” or “host” refers to either a human or non-humananimal.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

A “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

A “pharmaceutically-acceptable salt” refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds.

The term “single chain variable fragment or scFv” refers to an Fvfragment in which the heavy chain domain and the light chain domain arelinked. One or more scFv fragments may be linked to other antibodyfragments (such as the constant domain of a heavy chain or a lightchain) to form antibody constructs having one or more antigenrecognition sites.

As used herein, a “stress protein,” also known as a “heat shock protein”or “Hsp,” is a protein that is encoded by a stress gene, and istherefore typically produced in significantly greater amounts upon thecontact or exposure of the stressor to the organism. The term “stressprotein” as used herein is intended to include such portions andpeptides of a stress protein. A “stress gene,” also known as “heat shockgene”, as used herein, refers a gene that is activated or otherwisedetectably upregulated due to the contact or exposure of an organism(containing the gene) to a stressor, such as heat shock, hypoxia,glucose deprivation, heavy metal salts, inhibitors of energy metabolismand electron transport, and protein denaturants, or to certainbenzoquinone ansamycins. Nover, L., Heat Shock Response, CRC Press,Inc., Boca Raton, Fla. (1991). “Stress gene” also includes homologousgenes within known stress gene families, such as certain genes withinthe Hsp70 and Hsp90 stress gene families, even though such homologousgenes are not themselves induced by a stressor. Each of the terms stressgene and stress protein as used in the present specification may beinclusive of the other, unless the context indicates otherwise.

“Treating” a disease in a subject or “treating” a subject having adisease refers to subjecting the subject to a pharmaceutical treatment,e.g., the administration of a drug, such that the extent of the diseaseis decreased or prevented. Treatment includes (but is not limited to)administration of a composition, such as a pharmaceutical composition,and may be performed either prophylactically, or subsequent to theinitiation of a pathologic event.

The term “vaccine” refers to a substance that elicits an immune responseand also confers protective immunity upon a subject.

“Vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of preferredvector is an episome, i.e., a nucleic acid capable of extra-chromosomalreplication. Preferred vectors are those capable of autonomousreplication and/or expression of nucleic acids to which they are linked.Vectors capable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of “plasmids” which refer generally to circular doublestranded DNA loops, which, in their vector form are not bound to thechromosome. In the present specification, “plasmid” and “vector” areused interchangeably as the plasmid is the most commonly used form ofvector. However, as will be appreciated by those skilled in the art, theinvention is intended to include such other forms of expression vectorswhich serve equivalent functions and which become subsequently known inthe art.

1. Engineered Antibody-Stress Protein Fusion Polypeptides

Provided are fusion polypeptides comprising an engineered antibody and astress protein. The engineered antibody may comprise for example, atleast one scFv, at least one Fab fragment, at least one Fv fragment,etc. It may be monovalent or it may be multivalent. In embodimentswherein the engineered antibody is multivalent, it may be bivalent,trivalent, tetravalent, etc. The multivalent antibodies may bemonospecific or multispecific, e.g., bispecific, trispecific,tetraspecific, etc. The multivalent antibodies may be in any form, suchas a diabody, triabody, tetrabody, etc. In certain embodiments, theengineered antibody is a Tandab. The stress protein may comprise anystress protein. In certain embodiments, the stress protein comprisesHSP70, for example, Mycobacterium tuberculosis HSP70 or Mycobacteriumbovus HSP70. The full-length polypeptide sequences of Mycobacteriumtuberculosis HSP70 and Mycobacterium bovus HSP70 are depicted in FIG. 2as SEQ ID NOs: 1 and 2, respectively.

Further detail about engineered antibodies and stress proteins which maybe incorporated into the subject fusion polypeptides is provided below.

A. Engineered Antibodies

Natural antibodies are themselves dimers, and thus, bivalent. If twohybridoma cells producing different antibodies are artificially fused,some of the antibodies produced by the hybrid hybridoma are composed oftwo monomers with different specificities. Such bispecific antibodiescan also be produced by chemically conjugating two antibodies. Naturalantibodies and their bispecific derivatives are relatively large andexpensive to produce. The constant domains of mouse antibodies are alsoa major cause of the human anti-mouse antibody (HAMA) response, whichprevents their extensive use as therapeutic agents. They can also giverise to unwanted effects due to their binding of Fc-receptors. For thesereasons, molecular immunologists have been concentrating on theproduction of the much smaller Fab- and Fv-fragments in microorganisms.These smaller fragments are not only much easier to produce, they arealso less immunogenic, have no effector functions, and, because of theirrelatively small size, they are better able to penetrate tissues andtumors. In the case of the Fab-fragments, the constant domains adjacentto the variable domains play a major role in stabilizing the heavy andlight chain dimer. Accordingly, while full-length or nearly full lengthengineered antibodies may comprise the subject fusion polypeptides,smaller, single domain engineered antibodies (that may be multivalentand multispecific) are preferred for use in the fusion polypeptides.

The Fv-fragment is much less stable, and a peptide linker may thereforebe introduced between the heavy and light chain variable domains toincrease stability. This construct is known as a single chainFv(scFv)-fragment. A disulfide bond is sometimes introduced between thetwo domains for extra stability. Thus far, tetravalent scFv-basedantibodies have been produced by fusion to extra polymerizing domainssuch as the streptavidin monomer that forms tetramers, and toamphipathic alpha helices. However, these extra domains can increase theimmunogenicity of the tetravalent molecule.

Bivalent and bispecific antibodies can be constructed using onlyantibody variable domains. A fairly efficient and relatively simplemethod is to make the linker sequence between the V_(H) and V_(L)domains so short that they cannot fold over and bind one another.Reduction of the linker length to 3-12 residues prevents the monomericconfiguration of the scFv molecule and favors intermolecular V_(H)-V_(L)pairings with formation of a 60 kDa non-covalent scFv dimer “diabody”(Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90, 6444-6448). Thediabody format can also be used for generation of recombinant bispecificantibodies, which are obtained by the noncovalent association of twosingle-chain fusion products, consisting of the V_(H) domain from oneantibody connected by a short linker to the V_(L) domain of anotherantibody. Reducing the linker length still further below three residuescan result in the formation of trimers (“triabody”, about 90 kDa) ortetramers (“tetrabody”, about 120 kDa) (Le Gall et al., 1999, FEBSLetters 453, 164-168). For a review of engineered antibodies,particularly single domain fragments, see Holliger and Hudson, 2005,Nature Biotechnology, 23:1126-1136. All of such engineered antibodiesmay be used in the fusion polypeptides provided herein.

Other multivalent engineered antibodies that may comprise the subjectfusion polypeptides are described in Lu, et al., 2003, J. Immunol. Meth.279:219-232 (di-diabodies or tetravalent bispecific antibodies); USPublished Application 20050079170 (multimeric Fv molecules or“flexibodies”), and WO99/57150 and Kipriyanov, et al., 1999, J. Mol.Biol. 293:41-56 (tandem diabodies, or “Tandabs”).

An engineered antibody may specifically bind, e.g., to a tumor cellantigen of a cancer to be treated or prevented by the methods of thepresent invention. Such antigens include, but are not limited to, forexample, antigens of a human sarcoma cell or carcinoma cell, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acutelymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, or heavy chain disease cell.

Engineered antibodies may specifically bind other antigens, includingdisease-associated and/or viral antigens. An engineered antibody mayspecifically bind diseased and/or virally infected cells expressingantigen on their surface.

Infectious diseases that can be treated or prevented by the methods ofthe present invention are caused by infectious agents. Such infectiousagents or antigens derived therefrom, that may be targeted by anengineered antibody of the present invention, include, but are notlimited to, viruses, bacteria, fungi, and protozoa. The invention is notlimited to treating or preventing infectious diseases caused byintracellular pathogens but is intended to include extracellularpathogens as well. Many medically relevant microorganisms have beendescribed extensively in the literature, e.g., see C. G. A Thomas,Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entirecontents of which is hereby incorporated by reference.

Infectious viruses of both human and non-human vertebrates, includeretroviruses, RNA viruses and DNA viruses expressing antigen. Examplesof viral antigens include but are not limited to antigens of:Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g.vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebolaviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses);Bimaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the etiological agents of Spongiform encephalopathies, the agentof delta hepatitis (thought to be a defective satellite of hepatitis Bvirus), the agents of non-A, non-B hepatitis (class I=internallytransmitted; class 2=parenterally transmitted (i.e. Hepatitis C);Norwalk and related viruses, and astroviruses).

Retroviral antigens that may be targeted include antigens of both simpleretroviruses and complex retroviruses. The simple retroviruses includethe subgroups of B-type retroviruses, C-type retroviruses and D-typeretroviruses. An example of a B-type retrovirus is mouse mammary tumorvirus (MMTV). The C-type retroviruses include subgroups C-type group A(including Rous sarcoma virus (RSV), avian leukemia virus (ALV), andavian myeloblastosis virus (AMV)) and C-type group B (including murineleukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus(MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV),reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)). TheD-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simianretrovirus type 1 (SRV-1). The complex retroviruses include thesubgroups of lentiviruses, T-cell leukemia viruses and the foamyviruses. Lentiviruses include HIV-1, but also include HIV-2, SIV, Visnavirus, feline immunodeficiency virus (FIV), and equine infectious anemiavirus (EIAV). The T-cell leukemia viruses include HTLV-1, HTLV-II,simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV).The foamy viruses include human foamy virus (HFV), simian foamy virus(SFV) and bovine foamy virus (BFV).

Examples of antigens of RNA viruses that may be bound by an engineeredantibody include, but are not limited to, antigens of the following:members of the family Reoviridae, including the genus Orthoreovirus(multiple serotypes of both mammalian and avian retroviruses), the genusOrbivirus (Bluetongue virus, Eugenangee virus, Kemerovo virus, Africanhorse sickness virus, and Colorado Tick Fever virus), the genusRotavirus (human rotavirus, Nebraska calf diarrhea virus, murinerotavirus, simian rotavirus, bovine or ovine rotavirus, avianrotavirus); the family Picornaviridae, including the genus Enterovirus(poliovirus, Coxsackie virus A and B, enteric cytopathic human orphan(ECHO) viruses, hepatitis A virus, Simian enteroviruses, Murineencephalomyelitis (ME) viruses, Poliovirus muris, Bovine enteroviruses,Porcine enteroviruses, the genus Cardiovirus (Encephalomyocarditis virus(EMC), Mengovirus), the genus Rhinovirus (Human rhinoviruses includingat least 113 subtypes; other rhinoviruses), the genus Apthovirus (Footand Mouth disease (FMDV); the family Calciviridae, including Vesicularexanthema of swine virus, San Miguel sea lion virus, Feline picornavirusand Norwalk virus; the family Togaviridae, including the genusAlphavirus (Eastern equine encephalitis virus, Semliki forest virus,Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus,Venezuelan equine encephalitis virus, Western equine encephalitisvirus), the genus Flavirius (Mosquito borne yellow fever virus, Denguevirus, Japanese encephalitis virus, St. Louis encephalitis virus, MurrayValley encephalitis virus, West Nile virus, Kunjin virus, CentralEuropean tick borne virus, Far Eastern tick borne virus, Kyasanur forestvirus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus),the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosaldisease virus, Hog cholera virus, Border disease virus); the familyBunyaviridae, including the genus Bunyvirus (Bunyamwera and relatedviruses, California encephalitis group viruses), the genus Phlebovirus(Sandfly fever Sicilian virus, Rift Valley fever virus), the genusNairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep diseasevirus), and the genus Uukuvirus (Uukuniemi and related viruses); thefamily Orthomyxoviridae, including the genus Influenza virus (Influenzavirus type A, many human subtypes); Swine influenza virus, and Avian andEquine Influenza viruses; influenza type B (many human subtypes), andinfluenza type C (possible separate genus); the family paramyxoviridae,including the genus Paramyxovirus (Parainfluenza virus type 1, Sendaivirus, Hemadsorption virus, Parainfluenza viruses types 2 to 5,Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measlesvirus, subacute sclerosing panencephalitis virus, distemper virus,Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus(RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice);forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus,Ross river virus, Venezuelan equine encephalitis virus, Western equineencephalitis virus), the genus Flavirius (Mosquito borne yellow fevervirus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitisvirus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus,Central European tick borne virus, Far Eastern tick borne virus,Kyasanur forest virus, Louping III virus, Powassan virus, Omskhemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genusPestivirus (Mucosal disease virus, Hog cholera virus, Border diseasevirus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus of mice); the family Rhabdoviridae,including the genus Vesiculovirus (VSV), ChanBipura virus, Flanders-HartPark virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses,and two probable Rhabdoviruses (Marburg virus and Ebola virus); thefamily Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus,Human enteric corona virus, and Feline infectious peritonitis (Felinecoronavirus).

Illustrative DNA viral antigens include, but are not limited to antigensof the family Poxyiridae, including the genus Orthopoxvirus (Variolamajor, Variola minor, Monkey pox Vaccinia, Cowpox, Buffalopox,Rabbitpox, Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), thegenus Avipoxvirus (Fowlpox, other avian poxvirus), the genusCapripoxvirus (sheeppox, goatpox), the genus Suipoxvirus (Swinepox), thegenus Parapoxvirus (contagious postular dermatitis virus, pseudocowpox,bovine papular stomatitis virus); the family Iridoviridae (African swinefever virus, Frog viruses 2 and 3, Lymphocystis virus of fish); thefamily Herpesviridae, including the alpha-Herpesviruses (Herpes SimplexTypes 1 and 2, Varicella-Zoster, Equine abortion virus, Equine herpesvirus 2 and 3, pseudorabies virus, infectious bovinekeratoconjunctivitis virus, infectious bovine rhinotracheitis virus,feline rhinotracheitis virus, infectious laryngotracheitis virus) theBeta-herpesviruses (Human cytomegalovirus and cytomegaloviruses ofswine, monkeys and rodents); the gamma-herpesviruses (Epstein-Barr virus(EBV), Marek's disease virus, Herpes saimiri, Herpesvirus ateles,Herpesvirus sylvilagus, guinea pig herpes virus, Lucke tumor virus); thefamily Adenoviridae, including the genus Mastadenovirus (Human subgroupsA, B, C, D, E and ungrouped; simian adenoviruses (at least 23serotypes), infectious canine hepatitis, and adenoviruses of cattle,pigs, sheep, frogs and many other species, the genus Aviadenovirus(Avian adenoviruses); and non-cultivatable adenoviruses; the familyPapoviridae, including the genus Papillomavirus (Human papillomaviruses, bovine papilloma viruses, Shope rabbit papilloma virus, andvarious pathogenic papilloma viruses of other species), the genusPolyomavirus (polyomavirus, Simian vacuolating agent (SV-40), Rabbitvacuolating agent (RKV), K virus, BK virus, JC virus, and other primatepolyoma viruses such as Lymphotrophic papilloma virus); the familyParvoviridae including the genus Adeno-associated viruses, the genusParvovirus (Feline panleukopenia virus, bovine parvovirus, canineparvovirus, Aleutian mink disease virus, etc). Finally, DNA viralantigens may include viral antigens of viruses which do not fit into theabove families such as Kuru and Creutzfeldt-Jacob disease viruses andchronic infectious neuropathic agents.

Any of the above-described multivalent engineered antibodies may bedeveloped by one of skill in the art using routine recombinant DNAtechniques, for example as described in PCT International ApplicationNo. PCT/US86/02269; European Patent Application No. 184,187; EuropeanPatent Application No. 171,496; European Patent Application No. 173,494;PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Application No. 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw etal. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science229:1202-1207; Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No.5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; Beidler et al. (1988) J. Immunol.141:4053-4060; and Winter and Milstein, Nature, 349, pp. 293-99 (1991)).Preferably non-human antibodies are “humanized” by linking the non-humanantigen binding domain with a human constant domain (e.g. Cabilly etal., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.U.S.A., 81, pp. 6851-55 (1984)).

The antigen recognition sites or entire variable regions of theengineered antibodies may be derived from one or more parentalantibodies directed against any antigen of interest. The parentalantibodies can include naturally occurring antibodies or antibodyfragments, antibodies or antibody fragments adapted from naturallyoccurring antibodies, antibodies constructed de novo using sequences ofantibodies or antibody fragments known to be specific for an antigen ofinterest. Sequences that may be derived from parental antibodies includeheavy and/or light chain variable regions and/or CDRs, framework regionsor other portions thereof.

Multivalent, multispecific antibodies may contain a heavy chaincomprising two or more variable regions and/or a light chain comprisingone or more variable regions wherein at least two of the variableregions recognize different epitopes on the same antigen.

Candidate engineered antibodies for inclusion in the fusionpolypeptides, or the fusion polypeptides themselves, may be screened foractivity using a variety of known assays. For example, screening assaysto determine binding specificity are well known and routinely practicedin the art. For a comprehensive discussion of such assays, see Harlow etal. (Eds.), ANTIBODIES: A LABORATORY MANUAL; Cold Spring HarborLaboratory; Cold Spring Harbor, N.Y., 1988, Chapter 6.

Further provided are methods of selecting candidate engineeredantibodies. For example, candidates may be derived from human scFv andother antibody libraries. Accordingly, provided are antibody bacterialdisplay libraries. A library preferably comprises a plurality ofbacteria wherein the bacterial display, on average, has at least onecopy of a scFv or V_(H) or V_(L); the library comprises a plurality ofspecies of scFv or V_(H) or V_(L). In preferred embodiments, thebacterial display, on average, comprises at least 3, at least 4, or atleast 5 copies of a scFv or V_(H) or V_(L) per bacterium. Particularlypreferred libraries comprise, on average, at least about 10⁶, preferablyat least about 10⁷, more preferably at least about 10⁸ different speciesof scFv or V_(H) or V_(L). In a most preferred embodiment, theantibodies are encoded by a nucleic acid that is part of plasmid orphagemid vectors. In still yet another embodiment, this inventionprovides a nucleic acid library encoding as the bacterial displayantibody libraries. The nucleic acid library comprises at least about10⁶, more preferably at least about 10⁷, and most preferably at leastabout 10⁸ different plasmid or phagemid vectors.

Endocytosed bacteria can be selected by two different methods. One wayis to lyse and plate these mammalian cells on bacterial media containingappropriate antibiotic markers but such procedures are cumbersome andlaborious when a large library of >10⁸ variants has to be screened.Another approach is to express a fluorescent protein such as GFP in E.coli, and once endocytosed, the mammalian cell is fluorescent and can beisolated by FACS. GFP is a novel fluorescent marker to select forbacteria that are endocytosed because of the following features: a) GFPis a cytoplasmic protein with low toxicity (Chalfie et. al., Science263:802, 1994); therefore, the presence of GFP should have minimaleffects on the bacterial cell surface dynamics; b) GFP can becontinuously synthesized, which minimizes the effect offluorescence-signal dilution during bacterial replication; and c) GFP iseasily imaged and quantitated (Wang and Hazelrigg, Nature 369:400,1994). Furthermore, the fluorescence intensity of a single mammaliancell is directly proportional to the number of bacteria associated withit (Valdivia et. al., Gene 173:47, 1996). Therefore, flow cytometricanalysis of GFP-producing bacteria associated with host cells provides arapid and convenient measurement of bacterial adherence and invasion. Ithas been shown that a) the gene gfp is expressed and a functionalfluorescent GFP is produced in diverse bacterial systems such as E.coli, Yersinia pseudotuberculosis, Salmonella typhimurium, andMycobacterium marinum, b) production of GFP did not alter theinteraction of three pathogens with their respective host cells, c)intracellular bacterial pathogens producing GFP can be imaged inassociation with live cells and tissues, and d) GFP production can bedetected by flow cytometry and be used to measure the degree ofbacterial association with mammalian cells (Valdivia et. al. supra).

It is possible to directly select internalizing antibody candidates fromlarge non-immune or immune bacterial display libraries by recoveringinternalized bacteria from within mammalian cells afterreceptor-mediated endocytosis. Thus, in one embodiment, this inventionprovides methods of selecting polypeptide or antibody domains that areinternalized into specific target cells. The methods involve a)contacting one or more of target cells with one or more members of abacterial display library; b) culturing the target cells underconditions where members of the display library can be internalized; andc) identifying internalized members of the bacterial display library ifmembers of the bacterial display library are internalized into one ormore of the target cells. Preferably, the methods additionally involvecontacting members of the bacterial display library with cells ofsubtractive cell lines; and then washing the target cells to remove thecells of a subtractive cell line; and to remove members of the bacterialdisplay library that are non-specifically bound or weakly bound to thetarget cells. In a preferred embodiment, the bacterial display libraryis an antibody bacterial display library, more preferably an antibodybacterial display library displaying single chain antibodies (scFv), orthe variable domains of either light (V_(L)) or heavy (V_(H)) chains.

In a preferred embodiment, the identifying step comprises recoveringinternalized bacterium and repeating steps of the process again tofurther select for internalizing binding moieties. In one embodiment,the recovering step involves lysing the target cells to releaseinternalized bacterium, and sub-culturing the bacterium to producebacteria for a subsequent round of selection. The recovering step caninvolve recovering infective bacterium, and/or recovering a nucleic acidencoding a bacterial-displayed antibody and/or selection of bacteriaexpressing a selectable marker. The identifying step can involvedetecting expression of a reporter gene, detecting the presence orquantity of a particular nucleic acid, or selection of bacterium via aselectable marker. The identifying step can also involve sorting ofmammalian cells with internalized bacteria by FACS. In preferredmethods, the cells of a subtractive cell line are present in at least2-fold excess over the target cells. In preferred methods, the targetcell line is grown adherent to a tissue culture plate and co-incubatedwith the subtracting cell line in suspension in a single culture flask.In particularly preferred methods, the contacting with a subtractivecell line is performed at a temperature (for example, at 4° C.) lowerthan the internalization culture conditions (for example, at 37° C.). Inparticularly preferred embodiments, the bacteria express a selectablemarker and/or a reporter gene. Preferred selectable markers include, butare not limited to genes (or cDNAs) encoding fluorescent proteins (forexample, GFP), and a chromogenic gene or cDNA (for example, betalactamase, luciferase, and beta galactosidase). In certain embodiments,the target cells can include cells that over express a particularreceptor, members of a cDNA expression library, cells that over expressa chemokine receptor, cells of a transformed cell line, cellstransformed with a gene or cDNA encoding a specific surface targetreceptor. Suitable subtractive cell lines include, but are not limitedto normal human fibroblasts, normal human breast cells, pancreaticcells, and cardiomyocytes.

The cell-surface receptors involved in receptor-mediated endocytosis canbe identified de novo (Gao et. al., J. Immunol. Meth. 274:185, 2003). Inthe first step, through a subtractive approach, the tumor-specificinternalizing scFvs are isolated by sequentially exposing the scFvlibrary to various human cells and then finally to the desired celltype. As the next step, the selected scFvs are used as probes for thesubsequent identification of their cognate receptors byimmunoprecipitation, mass spectrometry and database searching. Based onthis procedures scFvs specific to transferrin receptor in prostate tumorcells, and α₃β₁ integrin present in pancreatic adenocarcinoma cells wereselected (Gao et. al., supra). Such a subtractive approach has beensuccessfully used to select internalizing receptors on human breast andpancreatic carcinoma cell lines (Fransson et. al., Cancer Lett. 208:235,2004) as well as on prostate carcinoma cells (Liu et. al., Cancer Res.64:704, 2004).

Accordingly, the methods of this invention may also be used to identifyinternalizing receptors. Identifying an endocytosing receptor presentonly in hepatocytes (liver cells) and not in any other cell types is onesuch example. The methods generally involve any of the methods foridentifying internalizing antibodies or polypeptides identified are usedto probe the original target cells, or different cells. As theinternalizing antibodies or polypeptides so bind, they permit isolationof the cell bearing the internalizing receptor and isolation of thereceptor and/or the receptor epitope itself. Thus, in one embodiment themethods involve a) contacting one or more of the target cells with oneor more members of a bacterial display library, b) optionally, butpreferably, contacting members of the bacterial display library withcells of a subtractive cell line, c) optionally, but preferably, washingthe target cells to remove said cells of a subtractive cell line and toremove members of the bacterial display library that arenon-specifically bound or weakly bound or weakly bound to said targetcells, d) culturing the cells under conditions where members of saidbacterial display library can be internalized if bound to aninternalizing marker, e) identifying internalized members of thebacterial display library if members of the bacterial display libraryare internalized into one or more of said target cells, f) contactingthe same or different target cells with the identified internalizedmembers of step (e) or members propagated therefrom, whereby the membersbind to the surface of said target cells. The method can further involveisolating a component of the same or different target cells to which themembers bind. In some methods the “identifying” step involves recoveringinternalized bacteria and repeating steps (a-e) to further select forinternalizing receptors. The contacting, washing, culturing, andidentifying steps are preferably performed as described herein, and thesubtractive cell lines include cardiomyocytes, normal and cancerousbreast cells.

Other protein display technologies may be used in the above-describedmethods. Modification of such methods to incorporate other displaytechnologies is well known to one of skill in the art. A review ofexemplary protein display technologies that may be used in the presentmethods is provided below.

Protein Display Technologies:

Antibody engineering plays a critical role in developing antibodytherapies with superior pharmacokinetic and pharmacodynamic properties(Burks et. al., Proc. Natl. Acad. Sci. USA 94:412, 1997; U.S. Pat. No.6,180,341). Directed evolution involves, first, the generation of arecombinant library of protein-expressing clones with randomizedsequences using molecular biology techniques, and second, the use ofscreening technologies for the isolation of protein variants thatexhibit the most enhanced activity. The screening of large librariesrequires a physical link among a gene, the protein it encodes, and thedesired function. Such a link can be established by using a variety ofin vivo display technologies that have proven to be invaluable (Wittrup,Nature Biotechnol. 18:1039, 2000; Hayhurst and Georgiou, Curr. Opin.Chem. Biol. 5:683, 2001).

Protein display technologies collectively represent one of the mostpowerful tools for protein engineering (Olsen et. al., Curr. Opin.Biotechnol. 11:331, 2000). For display purposes, a protein is fused tothe C or N terminus of a polypeptide sequence that targets the resultingchimera onto the surfaces of biological particles such as viruses,bacteria, and yeast. Libraries are typically screened for ligand bindingby a series of adsorption-desorption cycles by a process called“panning”. Panning has been used successfully to screen highly complexlibraries made by cloning the mammalian antibody repertoire anddisplaying it on phage (up to 10¹¹ clones). For somewhat less diverselibraries (up to 10⁹ clones), display on bacteria or yeast coupled withflow cytometry is a powerful tool for the discovery of proteins withexceptionally high ligand-binding affinities (Chen et. al., NatureBiotechnol. 19:537, 2001). Although the importance of displaytechnologies for protein engineering is undisputed, the need to anchorthe target polypeptide onto the surface of a biological particle imposesa number of limitations that can significantly reduce the diversity ofthe library relative to the totality of proteins that can be produced ina soluble form within the cell. First, protein display requires that theprotein of interest be expressed as either a C- or N-terminal fusion, aprocess that can adversely affect protein function and/or stability.Second, protein display is subject to biological constraints associatedwith protein export and presentation, which may compromise the viabilityof the virus or cell. Third, display can introduce screening artifactssuch as avidity effects in phages (O'Connell et. al., J. Mol. Biol.321:49, 2002).

Candidate engineered antibodies may selected through a combination ofprotein display technologies, particularly bacterial displaytechnology—involving the construction of anchored periplasmic expression(APEx) libraries in the bacterial periplasm as well as librariesexpressed in bacterial cytoplasm, such that the candidate engineeredantibodies are properly folded and functionally active underphysiologically reducing environments of the cytosol.

One approach that has been is the isolation of scFvs from phage displaylibraries followed by screening large numbers of clones for expressionin E. coli or function in mammalian cells (Lecerf et. al., Proc. Natl.Acad. Sci. USA 98:4764, 2001; Gennari et. al., J. Mol. Biol. 335:193,2004; Emadi et. al., Biochemistry 43:2871, 2004). Others have used thetwo-hybrid system to isolate engineered antibodies (Tes et. al., J. Mol.Biol. 317:85, 2002; Tanaka et. al., EMBO J. 22:1025, 2003), but thisdoes not allow for fine-tuning of the antibody biophysical propertiessuch as affinity and expression.

A: Phage Display Library:

Display on M13 bacteriophage is the oldest and the most widely usedprotein library-screening method (Marks et. al., J. Mol. Biol. 222:581,1991; Marks et. al., J. Biol. Chem. 267:16007, 1992; Rodi and Makowski,Curr. Opin. Biotechnol. 10:87, 1999). Phage antibody libraries havebecome an important resource for the development of therapeuticantibodies (Bradbury and Marks, J. Immunol. Meth. 290:29, 2004). Largenon-immune libraries serve as a single pot resource for the rapidgeneration of human MAbs (HuMAbs) to a wide range of self and non-selfantigens, including tumor growth factor receptors (Li et. al., CancerGene Ther. 8:555, 2001; Liu et. al., Cancer Res. 64:704, 2004). Most ofthe MAbs isolated from combinatorial libraries expressed on phage havebeen selected using purified antigens or peptides immobilized onartificial surfaces. This approach may select MAbs that do not recognizethe native protein in a physiological context, especially with largemolecular mass cell surface receptors. Attempts have been made to selectantigen in native conformation using either cell lysates (Parren et.al., J. Virol. 70:9046, 1996; Sanna et. al., Proc. Natl. Acad. Sci. USA92:6439, 1995; Sawyer et. al., J. Immunol. Meth. 204:193, 1997) orliving cells (Andersen et. al., Proc. Natl. Acad. Sci. USA 93:1820,1996; Osbourn et. al., Immunotechnol. 3:293, 1998). Because of theheterogeneity of the starting material, such approaches requireelaborate protocols including subtractive steps to avoid the selectionof irrelevant antibodies. The few successful selections performed onheterogenous material were generally done using small libraries fromimmunized sources. The use of immunized libraries limits the spectrum ofantigen specificities that can potentially be obtained from the samelibrary and typically yield murine antibodies. There are only threereports of successful selection on cells using large non-immunelibraries (de Kruif et. al., Proc. Natl. Acad. Sci. USA 92:3938, 1995;Marks et. al., Biotechnology 111:1145, 1993; Vaughan et. al., NatureBiotechnol. 14:309, 1996).

The step limiting the selection of binders from large naïve libraries bycell panning seems to be the relatively high background binding ofnon-specific phage and relatively low binding of specific phage(Becerril et. al., Biochem. Biophys. Res. Comm. 255:386, 1999; Pereiraet. al., J. Immunol. Meth. 203:11, 1997; Watters et. al., Immunotechnol.3:21, 1997). The low binding of specific phage is partially related tothe low concentration of a given binding phage in the polyclonalpreparation (approximately 1.6×10⁻¹⁷ M for a single member of a 10⁹library in a phage preparation of 1×10¹³ particles/ml). The lowconcentration simultaneously limits the efficiency of both subtractionof common binders and enrichment of specific binders. To overcome thislimitation, it was resorted to take advantage of normal cell surfacereceptor biology. Many receptors undergo endocytosis upon ligandbinding. It was hypothesized that enrichment ratios of specific binderscould be significantly increased by recovering endocytosed phageantibodies from the cytosol after stringent removal of non-specificphage from the cell surface (Poul et. al., J. Mol. Biol. 301:1149,2000).

B: Yeast Surface Display Library:

Yeast surface display (YSD; Boder and Wittrup, Nature Biotechnol.15:553, 1997) is another proven tool for protein engineering. In YSD,the protein of interest is expressed as a fusion with a yeast matingprotein, Aga2p, which is targeted to the yeast cell wall. Once expressedon the yeast surface, protein properties such as stability and affinity,can be quantitatively measured using fluorescently labeled reagents andflow cytometry. Further, libraries of mutants can be sorted for desiredproperties using fluorescent activated cell sorting (FACS). YSD has beensuccessfully applied to several facets of antibody engineering:isolation of novel Abs against specific antigens from a non-immune HuMAblibrary (Feldhaus et. al., Nature Biotechnol. 21:163, 2003); affinitymaturation resulting in the highest affinity antibody reported to date(Boder et. al., Proc. Natl. Acad. Sci. USA 97:10701, 2000); andstability and extracellular expression optimization (Shusta et. al.,Nature Biotechnol. 18:754, 2000). In addition, YSD is a useful tool fordomain-level analysis of an antibody's binding site (paratope), andengineering of functional antibodies (Colby et. al., J. Mol. Biol.342:901, 2004). In an attempt to identify a minimal antibody fragmentwith superior expression and intracellular function, YSD was used toengineer an intracellularly non-functional scFv into a functionalsingle-domain V_(L) antibody through affinity maturation and bindingsite analysis.

Not withstanding all these advantages, a potential shortcoming of theYSD platform for application to antibody engineering might arise fromthe difference in redox environment on the cell surface as compared tothe cytoplasm, where disulfide bonds do not stably form. MAbs containhighly conserved intradomain disulfide bonds in both the V_(H) and V_(L)domains that hold the β-sheet-forming framework residues in a rigidconformation. Disruption of these disulfide bonds perturbs the domainstructure, reducing protein stability (Ramm et. al., J. Mol. Biol.290:535, 1999). This presumably is responsible for the disparity betweencell surface expression and cytoplasmic expression levels for the scFv.Further, the expression of fusion proteins (for example, scFv) isgenerally cis-dominant; that is, the expression of the fusion protein isonly as good as the expression of the member with the lowest stability,so an alternative explanation of the improvement in expression observedwhen the V_(H) is eliminated is that the V_(H) domain of 2.4.3 wassignificantly less stable than the V_(L) under reducing conditions(Colby et. al., supra).

An important issue with any library screening technology (both phage andyeast display technologies) is the ability to express isolated clones ata high level. Existing display formats involve fusion to large anchoringsequences, which can influence the expression characteristics of thedisplayed proteins. For this reason, scFvs that display well as fusionsin phage, yeast, or bacteria (particularly the protein librariesexpressed on the outer membrane) may not necessarily be amenable to highexpression in soluble form as nonfusion proteins (Hayhurst et. al., J.Immunol. Meth. 276:185, 2003). In contrast, the short (6-aa) sequencerequired for N-terminal tethering of proteins onto the cytoplasmicmembrane in APEx display is unlikely to affect the expressioncharacteristics of the fusion. Consistent with this hypothesis, allthree affinity-enhanced clones to the anthrax PA toxin isolated by APExexhibited excellent soluble expression characteristics despite havingnumerous amino acid substitutions, suggesting that the isolation ofclones that can readily be produced in soluble form in bacteria on alarge scale may be an intrinsic feature of APEx selections (Harvey et.al., Proc. Natl. Acad. Sci. USA 101:9193, 2004).

C: APEx Bacterial Display Library:

A flow cytometry-based method has been developed using bacterialexpression for the efficient selection of high-affinity ligand-bindingproteins, and specifically scFvs, from combinatorial libraries. APEx isbased on the anchoring of proteins to the periplasmic side of the innermembrane, followed by disruption of the outer membrane before incubationwith fluorescently labeled antigen and FC sorting (Harvey et. al., Proc.Natl. Acad. Sci. USA 101:9193, 2004). In APEx, proteins are expressed inperiplasm by tethering to the inner membrane of E. coli. Afterchemical/enzymatic permeabilization of the bacterial outer membrane, E.coli cells expressing anchored scFv antibodies can be specificallylabeled with fluorescent antigens, ranging in size up to at least 240kDa, and analyzed by FC. Another advantage is that fusions between GFPand antigen can be expressed endogenously and captured byperiplasmically anchored scFv. Thus, after a washing step, cells thatexpress both the fluorescent antigen and an APEx-anchored scFv arehighly fluorescent and can be readily sorted from cells that expresseither only an scFv or GFP-antigen fusion alone.

With sorting rates of >400 million cells per hour, commercial FCmachines can be used to screen libraries of the size accessible withinthe constraints of microbial transformation efficiencies. Furthermore,multiparameter FC can provide valuable information regarding thefunction of each and every library clone in real time, thus helping toguide the library construction process and optimize sorting conditions(Daugherty, P. S. et. al., Proc. Natl. Acad. Sci. USA 97:2029, 2000). Inparticular, E. coli offers facile expression of recombinant protein andhigh DNA transformation efficiencies that allow for efficient largelibrary production and increased coverage of protein library sequencespace.

APEx display offers several advantages over previously developedbacterial periplasmic expression with cytometric screening method,called PECS (Chen et. al., Nature Biotechnol. 19:537, 2001), as well assurface display approaches such as phage and yeast display technologies:(i) APEX is an E. coli based system and therefore provides an easy routeto the creation of large libraries by transformation and preparativeprotein expression of isolated antibodies; (ii) by using a fattyacylated anchor to retain the protein in the inner membrane, a fusion asshort as 6 amino acids is all that is required for display. The shortfusion is unlikely to influence the affinity or expressioncharacteristics of the isolated proteins; (iii) the inner membrane lacksmolecules such as LPS or other complex carbohydrates that can stericallyinterfere with large antigen binding to displayed polypeptides; (iv) thefusion must only traverse one membrane before it is displayed, andtherefore biosynthetic limitations that might restrict the export ofcertain sequences to the yeast or bacterial surface may be circumvented;(v) display is accomplished by using either N- or C-terminal fusion,(vi) APEx can be used directly for proteins expressed from widely usedphage display vectors. Finally, (vii) APEx provides a means for thesimultaneous expression of fluorescent antigen and antibodies within thesame cell. This is particularly important for peptide antigens, andcircumvents time-consuming processes for synthesis, purification, andconjugation of preparative amounts of probe, as is required when thefluorescent antigen is incubated with the library. APEx can be used forthe detection of antigens ranging from small molecules (<1 kDa) tophycoerythrin conjugates (240 kDa), and possibly much larger antigens.

APEx display procedure can be used to derive a single domain antibodies(DAbs) from an scFv, when the binding energy of the scFv is contributedpredominantly by one of the two domains.

B. HSP70 Domains

Any suitable stress protein (heat shock protein (Hsp)) can be used inthe fusion polypeptides of the present invention. For example, Hsp60and/or Hsp70 can be used. Turning to stress proteins generally, cellsrespond to a stressor (typically heat shock treatment) by increasing theexpression of a group of genes commonly referred to as stress, or heatshock, genes. Heat shock treatment involves exposure of cells ororganisms to temperatures that are one to several degrees Celsius abovethe temperature to which the cells are adapted. In coordination with theinduction of such genes, the levels of corresponding stress proteinsincrease in stressed cells.

In bacteria, the predominant stress proteins are proteins with molecularsizes of about 70 and 60 kDa respectively, that are commonly referred toas Hsp70 and Hsp60. respectively. These and other specific stressproteins and the genes encoding them are discussed further below. Inbacteria, Hsp70 and Hsp60 typically represent about 1-3% of cell proteinbased on the staining pattern using sodium dodecyl sulfatepolyacrylamide gel electrophoresis and the stain Coomassie blue, butaccumulate to levels as high as 25% under stressful conditions. Stressproteins appear to participate in important cellular processes such asprotein synthesis, intracellular trafficking, and assembly anddisassembly of protein complexes. It appears that the increased amountsof stress proteins synthesized during stress serve primarily to minimizethe consequences of induced protein unfolding. Indeed, the preexposureof cells to mildly stressful conditions that induce the synthesis ofstress proteins affords protection to the cells from the deleteriouseffects of a subsequent more extreme stress.

The major stress proteins appear to be expressed in every organism andtissue type) examined so far. Also, it appears that stress proteinsrepresent the most highly conserved group of proteins identified todate. For example, when stress proteins in widely diverse organisms arecompared, Hsp90 and Hsp70 exhibit 50% or higher identity at the aminoacid level and share many similarities at non-identical positions. It isnoted that similar or higher levels of homology exist between differentmembers of a particular stress protein family within species.

The stress proteins, particularly Hsp70, Hsp60, Hsp20-30 and Hsp 10, areamong the major determinants recognized by the host immune system in theimmune response to infection by Mycobacterium tuberculosis andMycobacterium leprae. Young, R. A. and Elliott. T. J., Stress Proteins,Infection, And Immune Surveillance, Cell 50:5-8 (1989). Further, somerat arthritogenic T cells recognize Hsp60 epitopes. Van Eden, W. et al.,Nature 331:171-173 (1988). However, individuals, including healthyindividuals, with no history of mycobacterial infection or autoimmunedisease also carry T cells that recognize both bacterial and human Hsp60epitopes; a considerable fraction of T cells in healthy individuals thatare characterized by expression of the gamma-delta T cell receptorrecognize both self and foreign stress proteins. O'Brien, R. et al.,Cell 57:664-674 (1989). Thus, individuals, even healthy individualspossess T-cell populations that recognize both foreign and self stressprotein epitopes.

This system recognizing stress protein epitopes presumably constitutesan “early defense system” against invading organisms. Murray, P. J. andYoung, R. A., J. Bacteriol 174: 4193-6 (1992). The system may bemaintained by frequent stimulation by bacteria and viruses. As discussedbefore, healthy individuals have T cell populations recognizing selfstress proteins. Thus, the presence of autoreactive T cells iscompatible with normal health and does not cause autoimmune disease;this demonstrates the safety of stress proteins within an individual.The safety of stress proteins is additionally demonstrated by thesuccess and relative safety of BCG (Bacille Calmette Guerin, a strain ofMycobacterium bovis) vaccinations, which induce an immune responseagainst stress proteins that is also protective against Mycobacteriumtuberculosis.

Families of stress genes and proteins for use in the fusion polypeptidesare those well known in the art and include, for example, Hsp 100-200,Hsp100, Hsp90, Lon, Hsp70, Hsp60, TF55, Hsp40, FKBPs, cyclophilins,Hsp20-30, ClpP, GrpE, Hsp10, ubiquitin, calnexin, and protein disulfideisomerases. Macario, A. J. L., Cold Spring Harbor Laboratory Res.25:59-70, 1995; Parsell, D. A. & Lindquist, S. Ann. Rev. Genet.27:437-496 (1993); U.S. Pat. No. 5,232,833 (Sanders et al.). Aparticular group of stress proteins includes Hsp90. Hsp70. Hsp60,Hsp20-30, further preferably Hsp70 and Hsp60.

Hsp100-200 examples include Grp170 (for glucose-regulated protein).Grp170 resides in the lumen of the ER, in the pre-Golgi compartment, andmay play a role in immunoglobulin folding and assembly.

Hsp100 examples include mammalian Hsp110, yeast Hsp104, ClpA, ClpB,ClpC, ClpX and ClpY. Yeast Hsp104 and E. coli ClpA, form hexameric andE. coli ClpB, tetrameric particles whose assembly appears to requireadenine nucleotide binding. Clp protease provides a 750 kDaheterooligomer composed of ClpP (a proteolytic subunit) and of ClpA.ClpB-Y are structurally related to ClpA, although unlike ClpA they donot appear to complex with ClpP.

Hsp90 examples include HtpG in E. coli. Hsp83 and Hsc83 yeast, andHsp90alpha. Hsp90beta and Grp94 in humans. Hsp90 binds groups ofproteins, which proteins are typically cellular regulatory moleculessuch as steroid hormone receptors (e.g., glucocorticoid, estrogen,progesterone, and testosterone receptors), transcription factors andprotein kinases that play a role in signal transduction mechanisms.Hsp90 proteins also participate in the formation of large, abundantprotein complexes that include other stress proteins.

Lon is a tetrameric protein functioning as an ATP-dependent proteasedegrading non-native proteins in E. coli.

Hsp70 examples include Hsp72 and Hsc73 from mammalian cells, DnaK frombacteria, particularly mycobacteria such as Mycobacterium leprae,Mycobacterium tuberculosis, and Mycobacterium bovis (such asBacille-Calmette Guerin: referred to herein as Hsp71), DnaK fromEscherichia coli, yeast, and other prokaryotes, and BiP and Grp78. Hsp70is capable of specifically binding ATP as well as unfolded polypeptidesand peptides, thereby participating in protein folding and unfolding aswell as in the assembly and disassembly of protein complexes.

Hsp60 examples include Hsp65 from mycobacteria. Bacterial Hsp60 is alsocommonly known as GroEL, such as the GroEL from E. coli. Hsp60 formslarge homooligomeric complexes, and appears to play a key role inprotein folding. Hsp60 homologues are present in eukaryotic mitochondriaand chloroplasts.

TF55 examples include Tcpl, TRiC and thermosome. The proteins typicallyoccur in the cytoplasm of eukaryotes and some archaebacteria, and formmulti-membered rings, promoting protein folding. They are also weaklyhomologous to Hsp60.

Hsp40 examples include DnaJ from prokaryotes such as E. coli andmycobacteria and HSJ1, HDJ1 and Hsp40. Hsp40 plays a role as a molecularchaperone in protein folding, thermotolerance and DNA replication, amongother cellular activities.

FKBPs examples include FKBP12, FKBP13, FKBP25, and FKBP59, Fprl andNepl. The proteins typically have peptidyl-prolyl isomerase activity andinteract with immunosuppressants such as FK506 and rapamycin. Theproteins are typically found in the cytoplasm and the endoplasmicreticululum.

Cyclophilin examples include cyclophilins A, B and C. The proteins havepeptidyl-prolyl isomerase activity and interact with theimmunosuppressant cyclosporin A. The protein cyclosporin A bindscalcineurin (a protein phosphatase).

Hsp20-30 is also referred to as small Hsp. Hsp20-30 is typically foundin large homooligomeric complexes or, possibly, also heterooligomericcomplexes where an organism or cell type expresses several differenttypes of small Hsps. Hsp20-30 interacts with cytoskeletal structures,and may play a regulatory role in the polymerization/depolymerization ofactin. Hsp20-30 is rapidly phosphorylated upon stress or exposure ofresting cells to growth factors. Hsp20-30 homologues includealpha-crystallin.

ClpP is an E. coli protease involved in degradation of abnormalproteins. Homologues of ClpP are found in chloroplasts. ClpP forms aheterooligomeric complex with ClpA.

GrpE is an E. coli protein of about 20 kDa that is involved in both therescue of stress-damaged proteins as well as the degradation of damagedproteins. GrpE plays a role in the regulation of stress gene expressionin E. coli.

Hsp10 examples include GroES and Cpn10. Hsp10 is typically found in E.coli and in mitochondria and chloroplasts of eukaryotic cells. Hsp10forms a seven-membered ring that associates with Hsp60 oligomers. Hsp10is also involved in protein folding.

Ubiquitin has been found to bind proteins in coordination with theproteolytic removal of the proteins by ATP-dependent cytosolicproteases.

In particular embodiments, the stress proteins of the present inventionare obtained from enterobacteria, mycobacteria (particularly M. leprae,M. tuberculosis, M. vaccae, M. smegmatis and M. bovis), E. coli, yeast,Drosophila, vertebrates, avians, chickens, mammals, rats, mice,primates, or humans.

In particular embodiments e.g., in cases involving chemical conjugatesbetween a stress protein and an engineered antibody, the stress proteinsused are isolated stress proteins, which means that the stress proteinshave been selected and separated from the host cell in which they wereproduced. Such isolation can be carried out as described herein andusing routine methods of protein isolation known in the art.

The stress proteins may be in the form of acidic or basic salts, or inneutral form. In addition, individual amino acid residues may bemodified by oxidation or reduction. Furthermore, various substitutions,deletions, or additions may be made to the amino acid or nucleic acidsequences, the net effect of which is to retain or further enhance theincreased biological activity of the stress protein. Due to codedegeneracy, for example, there may be considerable variation innucleotide sequences encoding the same amino acid sequence. Portions ofstress proteins or peptides obtained from stress proteins may be used inthe fusion polypeptides, provided such portions or peptides include theepitopes involved with enhancing the immune response. Portions of stressproteins may be obtained by fragmentation using proteinases, or byrecombinant methods, such as the expression of only part of a stressprotein-encoding nucleotide sequence (either alone or fused with anotherprotein-encoding nucleic acid sequence). Peptides may also be producedby such methods, or by chemical synthesis. The stress proteins mayinclude mutations introduced at particular loci by a variety of knowntechniques, e.g., to enhance the effect on the immune system. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual. 2d Ed., ColdSpring Harbor Laboratory Press (1989); Drinkwater and Klinedinst Proc.Natl. Acad. Sci. USA 83:3402-3406 (1986); Liao and Wise, Gene 88:107-111(1990): Horwitz et al., Genome 3:112-117 (1989).

A fusion polypeptide may comprise an amino acid sequence that is atleast about 80%, 85%, 90%, 95%, 98%, or 99% identical to a stressprotein described herein.

2. Methods of Making the Engineered Antibody-Stress Protein FusionPolypeptides

Provided also are compositions and methods for making the engineeredantibody-stress protein fusion polypeptides. A fusion protein includingan engineered antibody and a stress protein can be produced byrecombinant means. For example, a nucleic acid encoding the stressprotein can be joined to either end of a nucleic acid sequence encodingthe engineered antibody such that the two protein-coding sequences aresharing a common translational reading frame and can be expressed as afusion protein including the engineered antibody and the stress protein.The combined sequence is inserted into a suitable vector chosen based onthe expression features desired and the nature of the host cell. In theexamples provided hereinafter, the nucleic acid sequences are assembledin a vector suitable for protein expression in the bacterium E. coli.Following expression in the chosen host cell, fusion protein can bepurified by routine biochemical separation techniques or byimmunoaffinity methods using an antibody to one or the other part of thefusion protein. Alternatively, the selected vector can add a tag to thefusion protein sequence, e.g., an oligohistidine tag as described in theexamples presented hereinafter, permitting expression of a tagged fusionprotein that can be purified by affinity methods using an antibody orother material having an appropriately high affinity for the tag.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory Press (1989); Deutscher, M. Guide to ProteinPurification Methods Enzymology, vol. 182. Academic Press, Inc. SanDiego, Calif. (1990). If a vector suitable for expression in mammaliancells is used. e.g., one of the vectors discussed below, the fusionprotein can be expressed and purified from mammalian cells.Alternatively, the mammalian expression vector (including fusionprotein-coding sequences) can be administered to a subject to directexpression of engineered antibody-stress protein fusion polypeptide inthe subject's cells. A nucleic acid encoding an engineeredantibody-stress protein fusion polypeptide can also be producedchemically and then inserted into a suitable vector for fusion proteinproduction and purification or administration to a subject. Finally, afusion protein can also be prepared chemically.

An isolated nucleic acid composition encoding the fusion polypeptide maycomprise a nucleotide sequence that is at least about 80%, 85%, 90%,95%, 98%, or 99% identical to a nucleotide sequence encoding a stressprotein described herein.

Techniques for making fusion genes are well known in the art.Essentially, the joining of various DNA fragments coding for differentpolypeptide sequences is performed in accordance with conventionaltechniques, employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene may be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments may be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which may subsequently be annealed to generate a chimeric genesequence (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al., John Wiley & Sons: 1992). Accordingly, provided is anisolated nucleic acid comprising a fusion gene of a gene encoding atleast one engineered antibody and a gene encoding at least one stressprotein.

The nucleic acid may be provided in a vector comprising a nucleotidesequence encoding an engineered antibody-stress protein fusionpolypeptide, and operably linked to at least one regulatory sequence. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. The vector's copy number,the ability to control that copy number and the expression of any otherprotein encoded by the vector, such as antibiotic markers, should beconsidered. Such vectors may be administered in any biologicallyeffective carrier, e.g., any formulation or composition capable ofeffectively transfecting cells either ex vivo or in vivo with geneticmaterial encoding a chimeric polypeptide. Approaches include insertionof the nucleic acid in viral vectors including recombinant retroviruses,adenoviruses, adeno-associated viruses, human immunodeficiency viruses,and herpes simplex viruses-1, or recombinant bacterial or eukaryoticplasmids. Viral vectors may be used to transfect cells directly; plasmidDNA may be delivered alone with the help of, for example, cationicliposomes (lipofectin) or derivatized (e.g., antibody conjugated),polylysine conjugates, gramicidin S, artificial viral envelopes or othersuch intracellular carriers. Nucleic acids may also be directlyinjected. Alternatively, calcium phosphate precipitation may be carriedout to facilitate entry of a nucleic acid into a cell.

The subject nucleic acids may be used to cause expression andover-expression of an engineered antibody-stress protein fusionpolypeptide in cells propagated in culture, e.g. to produce fusionproteins or polypeptides.

Provided also is a host cell transfected with a recombinant gene inorder to express an engineered antibody-stress protein fusionpolypeptide. The host cell may be any prokaryotic or eukaryotic cell.For example, an engineered antibody-stress protein fusion polypeptidemay be expressed in bacterial cells, such as E. coli, insect cells(baculovirus), yeast, insect, plant, or mammalian cells. In thoseinstances when the host cell is human, it may or may not be in a livesubject. Other suitable host cells are known to those skilled in theart. Additionally, the host cell may be supplemented with tRNA moleculesnot typically found in the host so as to optimize expression of thepolypeptide. Other methods suitable for maximizing expression of thefusion polypeptide will be known to those in the art.

A cell culture includes host cells, media and other byproducts. Suitablemedia for cell culture are well known in the art. A fusion polypeptidemay be secreted and isolated from a mixture of cells and mediumcomprising the polypeptide. Alternatively, a fusion polypeptide may beretained cytoplasmically and the cells harvested, lysed and the proteinisolated. A fusion polypeptide may be isolated from cell culture medium,host cells, or both using techniques known in the art for purifyingproteins, including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, and immunoaffinitypurification with antibodies specific for particular epitopes of afusion.

Thus, a nucleotide sequence encoding all or part of an engineeredantibody-stress protein fusion polypeptide may be used to produce arecombinant form of a protein via microbial or eukaryotic cellularprocesses. Ligating the sequence into a polynucleotide construct, suchas an expression vector, and transforming or transfecting into hosts,either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic(bacterial cells), are standard procedures. Similar procedures, ormodifications thereof, may be employed to prepare recombinant fusionpolypeptides by microbial means or tissue-culture technology in accordwith the subject invention.

An isolated nucleic acid composition encoding the fusion polypeptide maycomprise a nucleotide sequence that is at least about 80%, 85%, 90%,95%, 98%, or 99% identical to a nucleotide sequence encoding anengineered antibody described herein.

Expression vehicles for production of a recombinant protein includeplasmids and other vectors. For instance, suitable vectors for theexpression of a fusion polypeptide include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

In another embodiment, the nucleic acid is an engineered antibody-stressprotein fusion polypeptide operably linked to a bacterial promoter,e.g., the anaerobic E. coli, NirB promoter or the E. coli lipoproteinllp promoter, described, e.g., in Inouye et al. (1985) Nucl. Acids Res.13:3101; Salmonella pagC promoter (Miller et al., supra), Shigella entpromoter (Schmitt and Payne, J. Bacteriol. 173:816 (1991)), the tetpromoter on Tn10 (Miller et al., supra), or the ctx promoter of Vibriocholera. Any other promoter can be used. The bacterial promoter can be aconstitutive promoter or an inducible promoter. An exemplary induciblepromoter is a promoter which is inducible by iron or in iron-limitingconditions. In fact, some bacteria, e.g., intracellular organisms, arebelieved to encounter iron-limiting conditions in the host cytoplasm.Examples of iron-regulated promoters of FepA and TonB are known in theart and are described, e.g., in the following references: Headley, V. etal. (1997) Infection & Immunity 65:818; Ochsner, U. A. et al. (1995)Journal of Bacteriology 177:7194; Hunt, M. D. et al. (1994) Journal ofBacteriology 176:3944; Svinarich, D. M. and S. Palchaudhuri. (1992)Journal of Diarrhoeal Diseases Research 10:139; Prince, R. W. et al.(1991) Molecular Microbiology 5:2823; Goldberg, M. B. et al. (1990)Journal of Bacteriology 172:6863; de Lorenzo, V. et al. (1987) Journalof Bacteriology 169:2624; and Hantke, K. (1981) Molecular & GeneralGenetics 182:288.

A plasmid preferably comprises sequences required for appropriatetranscription of the nucleic acid in bacteria, e.g., a transcriptiontermination signal. The vector can further comprise sequences encodingfactors allowing for the selection of bacteria comprising the nucleicacid of interest, e.g., gene encoding a protein providing resistance toan antibiotic, sequences required for the amplification of the nucleicacid, e.g., a bacterial origin of replication.

In another embodiment, a signal peptide sequence is added to theconstruct, such that the fusion polypeptide is secreted from cells. Suchsignal peptides are well known in the art.

In one embodiment, the powerful phage T5 promoter, that is recognized byE. coli RNA polymerase is used together with a lac operator repressionmodule to provide tightly regulated, high level expression orrecombinant proteins in E. coli. In this system, protein expression isblocked in the presence of high levels of lac repressor.

In one embodiment, the DNA is operably linked to a first promoter andthe bacterium further comprises a second DNA encoding a first polymerasewhich is capable of mediating transcription from the first promoter,wherein the DNA encoding the first polymerase is operably linked to asecond promoter. In a preferred embodiment, the second promoter is abacterial promoter, such as those delineated above. In an even morepreferred embodiment, the polymerase is a bacteriophage polymerase,e.g., SP6, T3, or T7 polymerase and the first promoter is abacteriophage promoter, e.g., an SP6, T3, or T7 promoter, respectively.Plasmids comprising bacteriophage promoters and plasmids encodingbacteriophage polymerases can be obtained commercially, e.g., fromPromega Corp. (Madison, Wis.) and InVitrogen (San Diego, Calif.), or canbe obtained directly from the bacteriophage using standard recombinantDNA techniques (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning:A Laboratory Manual, Cold Spring Laboratory Press, 1989). Bacteriophagepolymerases and promoters are further described, e.g., in the followingreferences: Sagawa, H. et al. (1996) Gene 168:37; Cheng, X. et al.(1994) PNAS USA 91:4034; Dubendorff, J. W. and F. W. Studier (1991)Journal of Molecular Biology 219:45; Bujarski, J. J. and P. Kaesberg(1987) Nucleic Acids Research 15:1337; and Studier, F. W. et al. (1990)Methods in Enzymology 185:60). Such plasmids can further be modifiedaccording to the specific embodiment of the engineered antibody-stressprotein fusion polypeptide to be expressed.

In another embodiment, the bacterium further comprises a DNA encoding asecond polymerase which is capable of mediating transcription from thesecond promoter, wherein the DNA encoding the second polymerase isoperably linked to a third promoter. The third promoter may be abacterial promoter. However, more than two different polymerases andpromoters could be introduced in a bacterium to obtain high levels oftranscription. The use of one or more polymerases for mediatingtranscription in the bacterium can provide a significant increase in theamount of polypeptide in the bacterium relative to a bacterium in whichthe DNA is directly under the control of a bacterial promoter. Theselection of the system to adopt will vary depending on the specificuse, e.g., on the amount of protein that one desires to produce.

Generally, a nucleic acid encoding a fusion polypeptide is introducedinto a host cell, such as by transfection, and the host cell is culturedunder conditions allowing expression of the fusion polypeptide. Methodsof introducing nucleic acids into prokaryotic and eukaryotic cells arewell known in the art. Suitable media for mammalian and prokaryotic hostcell culture are well known in the art. Generally, the nucleic acidencoding the subject fusion polypeptide is under the control of aninducible promoter, which is induced once the host cells comprising thenucleic acid have divided a certain number of times. For example, wherea nucleic acid is under the control of a beta-galactose operator andrepressor, isopropyl beta-D-thiogalactopyranoside (IPTG) is added to theculture when the bacterial host cells have attained a density of aboutOD₆₀₀ 0.45-0.60. The culture is then grown for some more time to givethe host cell the time to synthesize the polypeptide. Cultures are thentypically frozen and may be stored frozen for some time, prior toisolation and purification of the polypeptide.

When using a prokaryotic host cell, the host cell may include a plasmidwhich expresses an internal T7 lysozyme, e.g., expressed from plasmidpLysSL (see Examples). Lysis of such host cells liberates the lysozymewhich then degrades the bacterial membrane.

Other sequences that may be included in a vector for expression inbacterial or other prokaryotic cells include a synthetic ribosomalbinding site; strong transcriptional terminators, e.g., to from phagelambda and t₄ from the rrnB operon in E. coli, to prevent read throughtranscription and ensure stability of the expressed polypeptide; anorigin of replication, e.g., ColE1; and beta-lactamase gene, conferringampicillin resistance.

Other host cells include prokaryotic host cells. Even more preferredhost cells are bacteria, e.g., E. coli. Other bacteria that can be usedinclude Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp.,Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp.,Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp.,Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp.,Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp.,Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp.Most of these bacteria can be obtained from the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209).

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al., (1983)in Experimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83). These vectors may replicate in E. coli due the presenceof the pBR322 ori, and in S. cerevisiae due to the replicationdeterminant of the yeast 2 micron plasmid. In addition, drug resistancemarkers such as ampicillin may be used.

In certain embodiments, mammalian expression vectors contain bothprokaryotic sequences to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and17. In some instances, it may be desirable to express the recombinantprotein by the use of a baculovirus expression system. Examples of suchbaculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal comprising pBlueBacIII).

In another variation, protein production may be achieved using in vitrotranslation systems. In vitro translation systems are, generally, atranslation system which is a cell-free extract comprising at least theminimum elements necessary for translation of an RNA molecule into aprotein. An in vitro translation system typically comprises at leastribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexesinvolved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex,comprising the cap-binding protein (CBP) and eukaryotic initiationfactor 4F (eIF4F). A variety of in vitro translation systems are wellknown in the art and include commercially available kits. Examples of invitro translation systems include eukaryotic lysates, such as rabbitreticulocyte lysates, rabbit oocyte lysates, human cell lysates, insectcell lysates and wheat germ extracts. Lysates are commercially availablefrom manufacturers such as Promega Corp., Madison, Wis.; Stratagene, LaJolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO/BRL, GrandIsland, N.Y. In vitro translation systems typically comprisemacromolecules, such as enzymes, translation, initiation and elongationfactors, chemical reagents, and ribosomes. In addition, an in vitrotranscription system may be used. Such systems typically comprise atleast an RNA polymerase holoenzyme, ribonucleotides and any necessarytranscription initiation, elongation and termination factors. An RNAnucleotide for in vitro translation may be produced using methods knownin the art. In vitro transcription and translation may be coupled in aone-pot reaction to produce proteins from one or more isolated DNAs.

When expression of a carboxy terminal fragment of a polypeptide isdesired, i.e. a truncation mutant, it may be necessary to add a startcodon (ATG) to the oligonucleotide fragment comprising the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position may be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al., (1987) J. Bacteriol 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al., (1987) PNAS USA 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, may beachieved either in vivo by expressing such recombinant polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al.).

In cases where plant expression vectors are used, the expression anengineered antibody-stress protein fusion polypeptide may be driven byany of a number of promoters. For example, viral promoters such as the35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature,310:511-514), or the coat protein promoter of TMV (Takamatsu et al.,1987, EMBO J., 6:307-311) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1994, EMBO J.,3:1671-1680; Broglie et al., 1984, Science, 224:838-843); or heat shockpromoters, e.g., soybean Hsp 17.5-E or Hsp 17.3-B (Gurley et al., 1986,Mol. Cell. Biol., 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plant virusvectors; direct DNA transformation; microinjection, electroporation,etc. For reviews of such techniques see, for example, Weissbach &Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press,New York, Section VIII, pp. 421-463; and Grierson & Corey, 1988, PlantMolecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

An alternative expression system which can be used to express apolypeptide tag or fusion protein comprising a polypeptide tag is aninsect system. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The PGHS-2 sequence maybe cloned into non-essential regions (for example the polyhedrin gene)of the virus and placed under control of an AcNPV promoter (for examplethe polyhedrin promoter). Successful insertion of the coding sequencewill result in inactivation of the polyhedrin gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedrin gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (e.g., see Smith et al., 1983, J. Virol., 46:584,Smith, U.S. Pat. No. 4,215,051).

In a specific embodiment of an insect system, the DNA encoding anengineered antibody-stress protein fusion polypeptide is cloned into thepBlueBacIII recombinant transfer vector (Invitrogen, San Diego, Calif.)downstream of the polyhedrin promoter and transfected into Sf9 insectcells (derived from Spodoptera frugiperda ovarian cells, available fromInvitrogen, San Diego, Calif.) to generate recombinant virus. Afterplaque purification of the recombinant virus high-titer viral stocks areprepared that in turn would be used to infect Sf9 or High Five™(BTI-TN-5B1-4 cells derived from Trichoplusia ni egg cell homogenates;available from Invitrogen, San Diego, Calif.) insect cells, to producelarge quantities of appropriately post-translationally modified subjectpolypeptide.

In other embodiments, an engineered antibody and stress protein areproduced separately and then linked, e.g. covalently linked, to eachother. For example, an engineered antibody and stress protein areproduced separately in vitro, purified, and mixed together underconditions under which the tag will be able to be linked to thepolypeptide of interest. For example, the stress protein and/or theengineered antibody can be obtained (isolated) from a source in which itis known to occur, can be produced and harvested from cell cultures, canbe produced by cloning and expressing a gene encoding the desired stressprotein or engineered antibody, or can be synthesized chemically.Furthermore, a nucleic acid sequence encoding the desired stress proteinor engineered antibody can be synthesized chemically. Such mixtures ofconjugated proteins may have properties different from single fusionproteins.

The present invention consists of both a non-chemical and chemicalmethod to link an engineered antibody to Mycobacterium TuberculosisHSP70 protein for the purpose of vaccination against the targetantigen(s) and/or cells expressing said target antigen(s). A fusionconstruct consisting of an antibody binding element and MycobacteriumTuberculosis HSP70 may be generated. The antibody binding element mayconsist for example of: Protein A, Protein G or Protein L or any proteinsegment(s) demonstrating high binding affinity for antibodies and/orscFvs. When needed, appropriate engineered antibodies such as scFV'scould be generated quickly, and stoichiometrically mixed with preformedPL-MTb HSP 70 fusion, creating the newly targeted scFv-PL-MTb HSP 70fusion vaccine.

Linkers (also known as “linker molecules” or “cross-linkers”) may beused to conjugate an engineered antibody and stress protein. Linkersinclude chemicals able to react with a defined chemical group ofseveral, usually two, molecules and thus conjugate them. The majority ofknown cross-linkers react with amine, carboxyl, and sulfhydryl groups.The choice of target chemical group is crucial if the group may beinvolved in the biological activity of the polypeptides to beconjugated. For example, maleimides, which react with sulfhydryl groups,may inactivate Cys-comprising peptides or proteins that require the Cysto bind to a target. Linkers may be homofunctional (comprising reactivegroups of the same type), heterofunctional (comprising differentreactive groups), or photoreactive (comprising groups that becomereactive on illumination).

Linker molecules may be responsible for different properties of theconjugated compositions. The length of the linker should be consideredin light of molecular flexibility during the conjugation step, and theavailability of the conjugated molecule for its target (cell surfacemolecules and the like.) Longer linkers may thus improve the biologicalactivity of the compositions of the present invention, as well as theease of preparation of them. The geometry of the linker may be used toorient a molecule for optimal reaction with a target. A linker withflexible geometry may allow the cross-linked polypeptides toconformationally adapt as they bind other polypeptides. The nature ofthe linker may be altered for other various purposes. For example, thearyl-structure of MBuS was found less immunogenic than the aromaticspacer of MBS. Furthermore, the hydrophobicity and functionality of thelinker molecules may be controlled by the physical properties ofcomponent molecules. For example, the hydrophobicity of a polymericlinker may be controlled by the order of monomeric units along thepolymer, e.g. a block polymer in which there is a block of hydrophobicmonomers interspersed with a block of hydrophilic monomers.

The chemistry of preparing and utilizing a wide variety of molecularlinkers is well-known in the art and many pre-made linkers for use inconjugating molecules are commercially available from vendors such asPierce Chemical Co., Roche Molecular Biochemicals, United StatesBiological, and the like.

3. Methods of Using the Engineered Antibody-Stress Protein FusionPolypeptides and Compositions Suitable Therefor

The engineered antibody-stress protein fusion polypeptides describedherein can be administered to a subject to enhance that subject's immuneresponse, particularly a cell-mediated cytolytic response, against acell expressing an antigen against which the engineered antibody domainsof the fusion polypeptide are directed. The fusion polypeptide maysimply enhance the immune response (thus serving as an immunogeniccomposition), or confer protective immunity (thus serving as a vaccine).

Thus, the engineered antibody-stress protein fusion polypeptidesproduced as described above may be purified to a suitable purity for useas a pharmaceutical composition. Generally, a purified composition willhave one species that comprises more than about 85 percent of allspecies present in the composition, more than about 85%, 90%, 95%, 99%or more of all species present. The object species may be purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single species. A skilled artisan may purifyan engineered antibody-stress protein fusion polypeptide using standardtechniques for protein purification, for example, immunoaffinitychromatography, size exclusion chromatography, etc. in light of theteachings herein. Purity of a polypeptide may be determined by a numberof methods known to those of skill in the art, including for example,amino-terminal amino acid sequence analysis, gel electrophoresis andmass-spectrometry analysis.

Accordingly, provided are pharmaceutical compositions comprising theabove-described engineered antibody-stress protein fusion polypeptides.In one aspect, provided are pharmaceutically acceptable compositionswhich comprise a therapeutically-effective amount of one or more of thecompounds described above, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Inanother aspect, in certain embodiments, the compounds may beadministered as such or in admixtures with pharmaceutically acceptablecarriers and may also be administered in conjunction with other agents.Conjunctive (combination) therapy thus includes sequential, simultaneousand separate, or co-administration of the active compound in a way thatthe therapeutic effects of the first administered one has not entirelydisappeared when the subsequent is administered.

The engineered antibody-stress protein fusion polypeptides describedherein can be administered to a subject in a variety of ways. The routesof administration include intradermal, transdermal (e.g., slow releasepolymers), intramuscular, intraperitoneal, intravenous, subcutaneous,oral, epidural and intranasal routes. Any other convenient route ofadministration can be used, for example, infusion or bolus injection, orabsorption through epithelial or mucocutaneous linings. In addition, thecompositions described herein can contain and be administered togetherwith other pharmacologically acceptable components such as biologicallyactive agents (e.g., adjuvants such as alum), surfactants (e.g.,glycerides), excipients (e.g., lactose), carriers, diluents andvehicles. Furthermore, the compositions can be used ex vivo as a meansof stimulating white blood cells obtained from a subject to elicit,expand and propagate antigen-specific immune cells in vitro that aresubsequently reintroduced into the subject.

Further, an engineered antibody-stress protein fusion polypeptide can beadministered by in vivo expression of a nucleic acid encoding suchprotein sequences into a human subject. Expression of such a nucleicacid can also be achieved ex vivo as a means of stimulating white bloodcells obtained from a subject to elicit, expand and propagateantigen-specific immune cells in vitro that are subsequentlyreintroduced into the subject. Expression vectors suitable for directingthe expression of engineered antibody-stress protein fusion polypeptidescan be selected from the large variety of vectors currently used in thefield. Preferred will be vectors that are capable of producing highlevels of expression as well as are effective in transducing a gene ofinterest. For example, recombinant adenovirus vector pJM17 (All et al.,Gene Therapy 1:367-84 (1994); Berkner K. L., Biotechniques 6:616-241988), second generation adenovirus vectors DE1/DE4 (Wang and Finer,Nature Medicine 2:714-6 (1996)), or adeno-associated viral vectorAAV/Neo (Muro-Cacho et al., J. Immunotherapy 11:231-7 (1992)) can beused. Furthermore, recombinant retroviral vectors MFG (Jaffee et al.,Cancer Res. 53:2221-6 (1993)) or LN, LNSX, LNCX, LXSN (Miller andRosman, Biotechniques 7:980-9 (1989)) can be employed. Herpes simplexvirus-based vectors such as pHSV1 (Geller et al., Proc. Nat'l Acad. Sci.87:8950-4 (1990) or vaccinia viral vectors such as MVA (Sutter and Moss.Proc. Nat'l Acad. Sci. 89:10847-51 (1992)) can serve as alternatives.

Frequently used specific expression units including promoter and 3′sequences are those found in plasmid CDNA3 (Invitrogen), plasmid AH5,pRC/CMV (Invitrogen), pCMU II (Paabo et al., EMBO J. 5:1921-1927(1986)), pZip-Neo SV (Cepko et al., Cell 37:1053-1062 (1984)) and pSRa(DNAX, Palo Alto, Calif.). The introduction of genes into expressionunits and/or vectors can be accomplished using genetic engineeringtechniques, as described in manuals like Molecular Cloning and CurrentProtocols in Molecular Biology (Sambrook, J., et al., Molecular Cloning,Cold Spring Harbor Press (1989); Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates andWiley-Interscience (1989)). A resulting expressible nucleic acid can beintroduced into cells of a human subject by any method capable ofplacing the nucleic acid into cells in an expressible form, for exampleas part of a viral vector such as described above, as naked plasmid orother DNA, or encapsulated in targeted liposomes or in erythrocyteghosts (Friedman, T., Science, 244:1275-1281 (1989); Rabinovich, N. R.et al., Science. 265:1401-1404 (1994)). Methods of transduction includedirect injection into tissues and tumors, liposomal transfection (Fraleyet al., Nature 370:111-117 (1980)), receptor-mediated endocytosis(Zatloukal et al., Ann. N.Y. Acad. Sci. 660:136-153 (1992)), andparticle bombardment-mediated gene transfer (Eisenbraun et al., DNA &Cell. Biol. 12:791-797 (1993)).

The amount of engineered antibody-stress protein fusion polypeptide(fused, conjugated or noncovalently joined as discussed before) in thecompositions of the present invention is an amount which produces aneffective immunostimulatory response in a subject. An effective amountis an amount such that when administered, it induces an immune response.In addition, the amount of engineered antibody-stress protein fusionpolypeptide administered to the subject will vary depending on a varietyof factors, including the engineered antibody and stress proteinemployed, the size, age, body weight, general health, sex, and diet ofthe subject as well as on its general immunological responsiveness.Adjustment and manipulation of established dose ranges are well withinthe ability of those skilled in the art. For example, the amount ofengineered antibody-stress protein fusion polypeptide can be from about1 microgram to about 1 gram, preferably from about 100 microgram toabout 1 gram, and from about 1 milligram to about 1 gram. An effectiveamount of a composition comprising an expression vector is an amountsuch that when administered, it induces an immune response against theantigen against which the engineered antibody is directed. Furthermore,the amount of expression vector administered to the subject will varydepending on a variety of factors, including the engineered antibody andstress protein expressed, the size, age, body weight, general health,sex, and diet of the subject, as well as on its general immunologicalresponsiveness. Additional factors that need to be considered are theroute of application and the type of vector used. For example, whenprophylactic or therapeutic treatment is carried out with a viral vectorcontaining a nucleic acid encoding an engineered antibody-stress proteinfusion polypeptide, the effective amount will be in the range of 10⁴ to10¹² helper-free, replication-defective virus per kg body weight,preferably in the range of 10⁵ to 10¹¹ virus per kg body weight and mostpreferably in the range of 10⁶ to 10¹⁰ virus per kg body weight.

Determination of an effective amount of fusion polypeptide for inducingan immune response in a subject is well within the capabilities of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

An effective dose can be estimated initially from in vitro assays. Forexample, a dose can be formulated in animal models to achieve aninduction of an immune response using techniques that are well known inthe art. One having ordinary skill in the art could readily optimizeadministration to humans based on animal data. Dosage amount andinterval may be adjusted individually. For example, when used as avaccine, the polypeptides and/or strains of the invention may beadministered in about 1 to 3 doses for a 1-36 week period. Preferably, 3doses are administered, at intervals of about 3-4 months, and boostervaccinations may be given periodically thereafter. Alternate protocolsmay be appropriate for individual patients. A suitable dose is an amountof polypeptide or strain that, when administered as described above, iscapable of raising an immune response in an immunized patient sufficientto protect the patient from the condition or infection for at least 1-2years.

The compositions may also include adjuvants to enhance immune responses.In addition, such proteins may be further suspended in an oil emulsionto cause a slower release of the proteins in vivo upon injection. Theoptimal ratios of each component in the formulation may be determined bytechniques well known to those skilled in the art.

Any of a variety of adjuvants may be employed in the vaccines of thisinvention to enhance the immune response. Most adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a specific or nonspecificstimulator of immune responses, such as lipid A, or Bortadellapertussis. Suitable adjuvants are commercially available and include,for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant(Difco Laboratories) and Merck Adjuvant 65 (Merck and Company, Inc.,Rahway, N.J.). Other suitable adjuvants include alum, biodegradablemicrospheres, monophosphoryl lipid A, quil A, SBAS1c, SBAS2 (Ling etal., 1997, Vaccine 15:1562-1567), SBAS7, Al(OH)₃ and CpG oligonucleotide(WO96/02555).

In the vaccines of the present invention, the adjuvant may induce a Th1type immune response. Suitable adjuvant systems include, for example, acombination of monophosphoryl lipid A, preferably 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) together with an aluminum salt. Anenhanced system involves the combination of a monophosphoryl lipid A anda saponin derivative, particularly the combination of 3D-MLP and thesaponin QS21 as disclosed in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol as disclosed inWO 96/33739. Previous experiments have demonstrated a clear synergisticeffect of combinations of 3D-MLP and QS21 in the induction of bothhumoral and Th1 type cellular immune responses. A particularly potentadjuvant formation involving QS21, 3D-MLP and tocopherol in anoil-in-water emulsion is described in WO 95/17210 and may comprise aformulation.

4. Kits

The present invention provides kits for expressing an engineeredantibody-stress protein fusion polypeptide. Such kits may be comprisedof nucleic acids encoding an engineered antibody-stress protein fusionpolypeptide. The nucleic acids may be included in a plasmid or a vector,e.g., a bacterial plasmid or viral vector. Other kits comprise anengineered antibody-stress protein fusion polypeptide. Furthermore, thepresent invention provides kits for producing and/or purifying anengineered antibody-stress protein fusion polypeptide.

The present invention provides kits for preventing or treatinginfectious, inflammatory, autoimmune or malignant disease in a patient.For example, a kit may comprise one or more pharmaceutical compositionsas described above and optionally instructions for their use. In stillother embodiments, the invention provides kits comprising one morepharmaceutical composition and one or more devices for accomplishingadministration of such compositions.

Kit components may be packaged for either manual or partially or whollyautomated practice of the foregoing methods. In other embodimentsinvolving kits, instructions for their use may be provided.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Example 1 Construction of a Mab-Hsp70 Conjugate and Immunization Usingthe Conjugate

A 14 amino acid long MISR2 peptide (SEQ ID NO 3: NANYSHLPPSGNRG) waschosen on the account of its stability, hydrophilicity and similaritywith mouse MISR2. The peptide was conjugated with HSP70 using 25%glutaraldehyde. A Balb/c mouse was immunized twice with 2-weeks intervalinto foot pads at 100 μg of MISR peptide-HSP70 conjugate. The immunelymph node cells were fused with sp2/0 myeloma cells. The supernatantwere screened by indirect ELISA using MISR peptide or MISR-HSP70 fusionprotein or pure HSP70. The positives were cloned 2-4 times andpropagated in mouse for ascites.

The antibodies were purified from ascitic fluids by doublesalt-precipitation using ammonium sulphate. The antibodies were testedin PAAG electrophoresis under denaturing condition (FIG. 3). Theantibodies were tested in indirect ELISA for binding with MISR-HSP70conjugate, MISR peptide or HSP70. The results are shown in FIG. 4.

Prophetic Example #1 Tandab Comprising scFv and HSP70

FIG. 1 depicts an exemplary engineered antibody-stress protein fusionpolypeptide comprising a tetravalent Tandab (engineered antibody) andHSP70 (stress protein). Tetravalent Tandabs may be preparedsubstantially as described in WO 99/57150, US20050089519 and Kipriyanov,et al., 1999, J. Mol. Biol. 293:41-56, all of which references areexpressly incorporated herein by reference. Briefly, the constructencoding the single chain molecule comprising four antibody variabledomains may additionally incorporate a stress protein gene, for example,HSP70. Alternatively, the single chain molecule comprising four antibodyvariable domains may be produced separately and then linked, e.g.covalently linked, to a stress protein such as HSP70.

Prophetic Example #2 Production of scFv in E. coli

E. coli strain GX6712 (F galk2 rspL cI857) and plasmid pGX8773 may beobtained from Genexcorp (Gaithersburg, Md.). The expression vectorpGX8773 successfully encodes a single chain antibody construct, fused tothe OmpA signal sequence, and contains an interdomain linker. The linkeris the flexible linker peptide of Trichoderma reesi. Expression vectorpLY3 encodes the scFV VH and VL genes fused to the OmpA signal sequence,with the VH and VL domains tethered by the linker. Expression vectorsutilize a hybrid OL/PR lamba promoter with protein expression initiatedby a temperature shift from 30° C. to 42° C. in E. coli GX6712.(Mallender & Voss, J. Biol. Chem (1994) 269:199-206)

The scFv may be expressed and then tethered to HSP70 separately, or thescFv may be incorporated into a fusion polypeptide, as describedthroughout the specification and in Example 3 below.

Prophetic Example #3 Production of Mycobacterium tuberculosis HSP70-scFv Fusion in E. coli

A fusion of Mycobacterium tuberculosis HSP 70 with a scFv may beproduced in E. coli as follows:

I. Characteristics of the E. coli Strain Producing Proteins scFv andDnaK (HSP70) M. tuberculosis.

The E. coli strain DLT 1270 was generated from DH 10A by virtue ofintegration of the gene lac 1 into the chromosome with D1-transduction.The genotype of DH 10A was as follows: DH 10B (ara D139 Δ(ara, leu) 7697Δ(lac)X74 galU galK rpsL deoR φ80lacZ DM15 endA1 nupG recA1 mcrA Δ(mrrhsdRMS mcrAN)). For a reference to DH 10B, see Grant, S., G., et al.Proc. Natl. Acad. Sci. USA (1990) 87:4645-4649.

II. Characteristics of the Plasmid

As a vector, the plasmid “QIAGEN” pQE-30 (“QIAGEN” Product Guide,www.qiagen.com) may be used.

III. Cloning of the Sequence of the scFv Gene of Interest in theRecombinant Vector dnaK

For cloning, a previously obtained vector pQE30-dnaK-Y may be used. Therecombinant plasmid pQE30-E711-dnaK produces the hybrid protein6HIS-E7(type 11)-dnaK by allowing expression of the protein dnaK fusedwith a sequence 6HIS at the N-terminus. Recombinant products withcorrect orientation have been identified using a restriction analysis.The scFv gene of interest (e.g., from vector pGX8773 described aboveand/or amplified using PCR) may be excised from the source byrestriction digest and cloned at the BamHI site with pQE30-dnaK-Yplasmid.

IV. Protocol for the Cultivation of the Strain Producing Proteins scFvand DnaK (HSP70) M. tuberculosis.

For the cultivation, the nutrient medium Luria-Bertani (LB) may beprepared in distilled water and its pH adjusted to 7.5 with NaOH orcitric acid. The media should be sterilized in an autoclave at 1 atm for40 min. When the medium has cooled down to 40° C., ampicillin may beadded aseptically to a final concentration of 50 μg/mL. The media withagar may be aseptically transferred to a Petri dish.

The producing strain may then be added to the freshly prepared media LBwith agar. The Petri dish may be placed in a thermostat and incubated at37° C. overnight to allow the culture to grow. For the preparation ofthe night culture, a desired amount of the LB media may be prepared in aconical flask. The media is transferred to the thermally resistantconical flasks so that the amount does not exceed ¼ of the flasks'volume. An isolated colony of E. coli may be transferred from the Petridish and seeded in the conical flask. The flasks are placed in athermoshaker and incubated overnight at 37° C. at 50 rev/min.

V. Fermentation of DLT1270-pQE30-scFv-dnaK

Synthesis of the hybrid peptide scFv-DnaK may be induced by adding IPTGto the culture. The overnight culture DLT1270/pQE30-scFv-DnaK, grown inLB-media, may be diluted 1:100 and grown in a LB-media to OD600=0.5. 0.1mM IPTG is added and cultivation continued for 3 h. The density of theculture may be measured using a spectrophotometer. After the end offermentation, the biomass may be collected by centrifugation at 300×Gfor 15 minutes at 4° C. Generation of the protein may be followed byelectrophoresis in polyacrylamide gel.

Example 4 Production of MTBhsp70

MTBhsp70 was subcloned into the expression vector pET45b(+) by firstmodifying the vector to introduce the desired restriction site SfiI.This modification allows introduction of the MTBhsp70 protein at theNotI/XhoI site and other proteins such as scFvs, antigens, etc. at theSfiI/NotI site (FIG. 5). This particular approach can be modified tointroduce the desired proteins at the C-terminal of MTBhsp70. Using theMTBhsp70 plasmid provided by Dr. Richard Young, restriction sites NotIand XhoI were introduced at the 5′ and 3′ end respectively (FIG. 6).

Digestion of the amplified MTBhsp70 fragment shown in lane 1 of FIG. 2with the restriction enzymes NotI and XhoI unexpectedly revealed 2 bands(FIG. 7). Sequencing analyses revealed that MTBhsp70 contains internalNotI and SfiI restriction sites. These were removed using the strategydepicted in FIG. 8. The resulting MTBhsp70 pET-45b(+) construct was thenused to transform competent BL21(DE3) bacteria. Expression of MTBhsp70was induced by adding 1 mM IPTG. Cells were grown in LB medium at 37° C.to an OD₆₀₀ of 0.5. Cells were spun down and suspended in LB mediumcontaining 1 mM IPTG and growth continued for 4 hours at the indicatedtemperature. Cells were fractionated and aliquots were run on SDS-PAGEand proteins were stained with Coomassie Blue. When induced cells weregrown at 37° C. the majority of the MTBhsp70 protein was found ininsoluble inclusion bodies. By reducing the growth temperature postinduction to 30° C., large amounts of soluble MTBhsp70 were produced.The MTBhsp70 protein found in the soluble and periplasmic fractions ofBL21(DE3) grown at 30° C. was successfully purified by metal affinitychromatography (MAC) using Cobalt spin columns. Cells were grown at 37°C. to an OD₆₀₀ of 0.5 and then spun down. Cells were suspended in growthmedia containing 1 mM IPTG and allowed to grow for 4 hours at 30° C.Cells were fractionated according to standard methods consisting ofsolubilization with B-PER reagent from Pierce.

Example 5 Production of MTBhsp70-Fusion Proteins

In order to demonstrate the immunostimulatory properties ofMTBhsp70-fusion proteins, an Ovalbumin peptide-MTBhsp70 and twoscFv-MTBhsp70 fusion products were constructed.

I Ova-257-264-MTBhsp 70 fusion protein. Ovalbumin's immunodominantpeptide consisting of residues 257-264 (SIINFEKL) was fused to theN-terminal region of MTBhsp70 by digesting the MTBhsp70 pET-45b(+)plasmid with SfiI and NotI and introducing a linker coding for theimmunodominant peptide that is also digested with SfiI and NotI (FIG.9). Upon ligation, a number of colonies were obtained, and theiridentities were confirmed by sequencing (FIG. 10). As observed withMTBhsp70, induction of Ova-257-264-MTBhsp70 is optimum when the growthtemperature, post-IPTG induction, is kept at 30° C. Successfulexpression of Ova254-264-MTBhsp70 was obtained in the soluble fractionof BL21 (DE3).

IL scFv-MTBhsp 70 fusion proteins. scFvs were fused to the N-terminal ofMTBhsp70. A human combinatorial scFv phage display library wasconstructed and used it to select Ovalbumin specific scFvs. The otherscFv, MOV18, is specific for the high affinity Folate Receptor expressedon ovarian cancer cells. The cloning method is similar to the approachused for the introduction of the Ova₂₅₄₋₂₆₄ peptide at the N-terminal ofMTBhsp70. The SfiI/NotI scFv portion was purified from their respectiveplasmids followed by ligation into the SfiI/NotI digested expressionvector MTBhsp70 pET-45b(+). The anti-Ovalbumin scFvs had severalnon-sense mutations that had to be removed by site directed mutagenesis.However, upon induction of bacteria carrying both constructs, it wasfound that induction with IPTG resulted in the fusion proteins beingexpressed in inclusion bodies.

Example 6 Generation of Single Chain Variable Fragments (scFV) TargetingNew Antigen Linked to MTB Hsp70: a Novel Fusion Vaccine.

The generation of scFVs that bind to either known or uncharacterizedantigens by “panning” against the desired antigen may be carried out asfollows. The scFV is comprised of the antigen-binding domains of heavyV_(H) and light V_(K), chains of antibodies, and can be made in E. coli.Repertoires of these scFV combinations can be built from the genes ofheavy V_(H) and light V_(K) chains obtained from B-lymphocytes eitherbefore or after immunization, or from the V-gene segments that have beencloned and rearranged in vitro. These gene segments can becombinatorially cloned into an appropriate phagemid vector to generatean scFv library that can be used to transform E. coli. Infection ofthese transformed bacteria with a suitable helper phage results in theproduction of recombinant phages expressing scFvs on their surface.Using successive rounds of selection (panning) it is possible to enrichthe population of phage expressing scFvs with binding affinities to aparticular antigen. One can derive scFV's from naive or non-immunizedhuman lymphocytes, by combining elements of both immunized andnon-immunized lymphocytes, or by various combinations of the above witha synthetic antibody library generated through the introduction ofrandomized sequences in the antibody binding site. This method has beenused to rapidly generate scFV's against highly dangerous pathogens suchas SARS-associated coronavirus (SARS-Cov). The use of human B cells asthe source the V-gene segments has also eliminated the antigenicity ofscFV's.

The approach described herein may be used for targeting antigens whosesequence may not be known or structure even identified. In place ofdirectly linking synthetic antigen peptides to MTb HSP 70 to generate avaccine, instead scFV's may be linked to MTb HSP 70 to develop a novelfusion protein vaccine for presenting antigen to APCs in order togenerate both a humoral and CD-8 response. The scFV's can be selectedfor their binding to an uncharacterized antigen or to a characterizedantigen by binding studies, and the appropriate V-genes can then beselected. The scFV genes are fused to the gene for MTb HSP 70, and thenew fusion gene is expressed as the fusion protein in E. coli. This hasalready been successfully done, using the scFV for cholera toxin subunitB bound to MTb HSP 70. This scFV-MTb HSP 70 fusion protein bound toantigen with a binding affinity of approximately 10⁻⁹ M.

Example 7 Rapid Assembly of Novel Vaccine Using Pre-formed MTb-HSP 70with Linkers for scFvs

Through unique cloning strategies, large amounts of pure MTb HSP 70 maybe expressed with great efficiency. The scFV-MTb HSP 70 fusion vaccinemay be produced by cloning the individual scFV sequence, a linkersequence, and the MTb HSP 70 sequence into a phagemid vector forexpression of the entire fusion vaccine in E. coli. A fast method may beused for generating a new fusion vaccine in which previously produced(potentially GMP) MTb HSP 70 with a linker that preferentially binds toscFvs could be stockpiled, and then rapidly coupled to newly producedscFV's. This would dramatically reduce the complexity and time ofgenerating a new vaccine. Protein L from Peptostreptococcus magnuspreferentially binds to the backbone of the variable region of lightchains, leaving the antigen combining site unobstructed. The gene codingfor Protein L could be cloned into the plasmid coding the gene for MTbHSP 70 in order to create a fusion of Protein L (PL) and MTb HSP 70.Large amounts of this new fusion protein could be grown in advance andstored. When needed, appropriate scFV's could be generated quickly, andstoichiometrically mixed with preformed PL-MTb HSP 70 fusion, creatingthe newly targeted scFv-PL-MTb HSP 70 fusion vaccine.

An even simpler chemical technique that binds scFV's and Fab's as achemical linker may be used to further facilitate the process. Thus, theMTB HSP 70 fusion subunit, the MTb HSP 70—linker subunit, and scFV's maybe produced. An scFv-linker MTB HSP 70 fusion vaccine may be produced.Thereafter small changes in the antigen recognition moiety of the newscFV to deal with new pathogens might be made.

I. Expression of the recombinant heat shock protein 70 fromMycobacterium tuberculosis (MTB hsp70). Our rapid vaccine developmentstrategy relies on the availability of pure MTBhsp70 and the productionof pure protein/peptide-MTBhsp70 fusions. This was accomplish bysubcloning MTBhsp70 from the pKS11his vector obtained from Dr. RichardYoung at the Massachusetts Institute of Technology into the expressionvector pET45b(+) (Novagen). This vector is particularly useful as itadds a polyhistidine-tag at the N-terminal of the construct thusfacilitating protein purification by Nickel or Cobalt affinitychromatography. Furthermore, upon purification, the polyhistidine-tagcan be removed by enterokinase digestion. Our strategy consisted ofmodifying the vector such that an scFv-MTBhsp70 fusion construct couldbe obtained by taking advantage of the fact that scFvs contain uniqueSfiI/NotI restriction sites. We first modified pET45b(+) by introducingunique SfiI and NotI sites and subcloning MTBhsp70 into the NotI/XhoIsite of the modified vector. The Modified pET45b(+) expression vectorallows the insertion of scFv or protein or peptide antigens in theSfiI/NotI cloning region. An enterokinase recognition site is located atthe junction between PshAI and SfiI.

BL21 (DE3) cells were transformed with our modified vector andproduction of soluble MTBhsp70 may be induced upon addition of IPTG tothe growth media. Passage of the soluble cell extract over a Cobaltaffinity matrix yielded essentially pure MTBhsp70.

II. Making human scFv (single strain variable fragment) phage displaylibrary from peripheral lymphocytes of unimmunized donors. Total RNA wasprepared from peripheral blood lymphocytes (PBLs) of unimmunized donorsusing Ambion's Tri Reagent. PBLs were separated from red blood cells bycentrifugation through Ficoll-Hypaque, recovered and washed twice withPBS. 1 ml of Tri Reagent per 10×10⁶ PBLs. We typically observed yieldsof 50 to 70 micrograms of RNA per 18 ml of blood collected in EDTAcoated tubes.

Primer design. Primers originally described by Gregory Winter were used(Marks J D, Hoogenboom H R, Bonnert T P, McCafferty J, Griffiths A D,Winter G. By-passing immunization. Human antibodies from V-genelibraries displayed on phage. J Mol Biol 1991; 222581-97.). The forwardprimers were designed to match each member of the human V-gene families,and reverse primers were designed to match of the human germ line Jsegments. In addition, sets of PCR primers were designed to optimize therandom linking of V_(H) segments to V_(κ) or V_(λ) gene segments.Finally, a set of primers containing the desired restriction sites SfiIand NotI was used to reamplify the scFv gene repertoire to allow cloninginto the vector pCANTAB5E.

First-strand cDNA synthesis and Amplification of VHg, VHm, VI and Vk.Four first-strand cDNAs were synthesized from total RNA (SuperScript111enzyme, Invitrogen) using either IgG or IgM constant region-specificprimer for the heavy chains, or light chain κ or λ. constantregion-specific primers. These cDNAs were used to generate 4 separaterepertoires of scFv genes (V_(H)μ-Vκ, V_(H)μ-Vλ, V_(H)Y-Vκ, andV_(H)Y-Vλ).

Making scFv linker DNA. To make the scFv linker DNA, 52 separate PCRreactions were performed using each of the 4 forward J-segment-specificprimers from heavy chain in combination with each of the 13 reverse Vκand Vλ specific primers. The rearranged V_(H) and V_(L) PCR productswere combined with linker DNA overlapping the C termini of V_(H) and theN termini of V_(L) gene segments and subjected to PCR amplification. Theresulting scFv gene repertoires were subsequently amplified with primerscontaining SfiI and NotI restriction sites. The amplified scFv generepertoires (IgG-V_(L) and IgM-V_(L) repertoires) were digested withSfiI and NotI restriction enzymes and ligated into the similarlydigested phagemid vector pCANTAB 5E. The ligated scFvs were used totransform electrocompetent TG1 cells to yield a library of approximately1×10⁶ clones. Addition of CT helper phage to the transformed TG1 cellsyielded the scFvs phage library used for screening.

Construction of CT Helper phage. In order to minimize the production ofundesired helper phage, we constructed the infection deficient CT helperphage described by Kramer (Kramer, Cox et al. 2003). In order to rescuethe phage library, the CT-phage genome is packaged in a phage envelopecontaining a functional gene 3. This was accomplished by transformingelectrocompetent XL 1-Blue cells carrying the fully functional gene 3 onpUC19.

Non-chemical conjugation of target specific immunoglobulins to MTBhsp70.To take advantage of the availability of monoclonal antibodies directedagainst antigens of interest for vaccine development we intend to clonethe immunoglobulin binding region from Peptostreptococcus Magnus ProteinL (PpL) into the SfiI/NotI site of MTBhsp70 pET45b(+) vector. PpL is a719 amino acids protein with a series of 4 to 5 immunoglobulin lightchain binding repeated binding region (B1-B5)(Kastem W, Sjobring U,Bjorck L. Structure of peptostreptococcal protein L and identificationof a repeated immunoglobulin light chain-binding domain. J Biol Chem1992; 267:12820-5.). The B1-B5 repeated region of PpL may be amplifiedfrom EcoRI digested P. Magnus genomic DNA using primers LP 1 and LP2modified to include the SfiI and NotI restriction sites respectively(Kastem, Sjobring et al. 1992). This novel construct will take advantagethe light chain binding activity of proteinL without interfering withthe antigen binding property of the bound immunoglobulin. Conjugation ofa PpL affinity ligand mimic to MTBhsp70. An affinity ligand that mimicsthe immunoglobulin binding properties of protein L may be prepared asdescribed by Lowe (Roque A C, Taipa M A, Lowe C R. An artificial proteinL for the purification of immunoglobulins and fab fragments by affinitychromatography. J Chromatogr A 2005; 1064:157-67.). A stable conjugatethat could be used to bind scFv and behave similarly to protein L may begenerated.

EQUIVALENTS

The present invention provides, among other things, engineeredantibody-stress protein fusion polypeptides. While specific embodimentsof the subject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The appended claims are not intended to claim all suchembodiments and variations, and the full scope of the invention shouldbe determined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations.

REFERENCES

Incorporated by reference in their entirety are any polynucleotide andpolypeptide sequences which reference an accession number correlating toan entry in the public database of the National Center for BiotechnologyInformation (NCBI) on the world wide web at ncbi.nlm.nih.gov. Thecontents of all cited references including literature references, issuedpatents, published or non published patent applications as citedthroughout this application are hereby expressly incorporated byreference. Additionally, the following references are expresslyincorporated herein by reference:

-   1. Hoogenboom H R. Selecting and screening recombinant antibody    libraries. Nat Biotechnol 2005; 23:1105-16.-   2. Marks J D, Hoogenboom H R, Bonnert T P, McCafferty J, Griffiths A    D, Winter G. By-passing immunization. Human antibodies from V-gene    libraries displayed on phage. J Mol Biol 1991; 222581-97.-   3. Flego M, Di Bonito P, Ascione A, et al. Generation of human    antibody fragments recognizing distinct epitopes of the    nucleocapsid (N) SARSCoV protein using a phage display approach. BMC    Infect Dis 2005; 5:73.-   4. Duan J, Yan X, Guo X, et al. A human SARS-CoV neutralizing    antibody against epitope on S2 protein. Biochem Biophys Res Commun    2005; 33311 86-93.-   5. Zugel U, Kaufmann S H. Role of heat shock proteins in protection    from and pathogenesis of infectious diseases. Clin Microbiol Rev    1999; 12: 19-39.-   6. Srivastava P K, Maki R G. Stress-induced proteins in immune    response to cancer. Curr Top Microbiol Immunol 1991; 167:109-23.-   7. Chen W, Syldath U, Bellmann K, Burkart V, Kolb H. Human 60-kDa    heatshock protein: a danger signal to the innate immune system. J    Immunol 1999; 1621321 2-9.-   8. Kastern W, Sjobring U, Bjorck L. Structure of peptostreptococcal    protein L and identification of a repeated immunoglobulin light    chain-binding domain. J Biol Chem 1992; 267:12820-5.-   9. Roque A C, Taipa M A, Lowe C R. An artificial protein L for the    purification of immunoglobulins and fab fragments by affinity    chromatography. J Chromatogr A 2005; 1064:157-67.

1. A fusion polypeptide comprising an engineered antibody and a stressprotein.
 2. The fusion polypeptide of claim 1, wherein the engineeredantibody comprises at least one scFv.
 3. The fusion polypeptide of claim1, wherein the engineered antibody comprises at least one Fab fragment.4. The fusion polypeptide of claim 1, wherein the stress protein isHSP70.
 5. The fusion polypeptide of claim 4, wherein the stress proteinis Mycobacterium tuberculosis HSP70.
 6. The fusion polypeptide of claim4, wherein the stress protein is Mycobacterium bovus HSP70.
 7. Thefusion polypeptide of claim 1, wherein the engineered antibody ismultivalent.
 8. The fusion polypeptide of claim 7, wherein themultivalent engineered antibody is multispecific.
 9. The fusionpolypeptide of claim 7, wherein the engineered antibody is tetravalent.10. The fusion polypeptide of claim 9, wherein the engineered antibodyis a Tandab.
 11. The fusion polypeptide of claim 9, wherein the stressprotein is HSP70.
 12. The fusion polypeptide of claim 11, wherein thestress protein is Mycobacterium tuberculosis HSP70.
 13. The fusionpolypeptide of claim 11, wherein the stress protein is Mycobacteriumbovus HSP70.
 14. An isolated nucleic acid encoding the fusionpolypeptide of claim
 1. 15. An expression vector comprising the nucleicacid of claim
 14. 16. A cell comprising the expression vector of claim15.
 17. A pharmaceutical composition comprising an effective amount of afusion polypeptide of claim 1, and a pharmaceutically acceptablecarrier.
 18. An immunogenic composition or vaccine comprising a fusionpolypeptide of claim
 1. 19. A kit comprising a composition of claim 1.20. The kit of claim 19, further comprising instructions for use of thecomposition.